Reactive Oxygen Species and Oncoprotein Signaling-A Dangerous Liaison

Cellular antioxidant defense systems

With ROS generation on one side of the coin, ROS detoxification on the other is, therefore, crucial to keep the intracellular ROS level in check. To this end, several antioxidant enzymes have been shown to detoxify intracellular ROS (97, 100). These enzymatic systems include SOD, catalase, glutathione peroxidase (GPx), glutathione reductase (GR), Prx, and Trx. SOD comprises three isoforms: SOD1 or Cu/Zn-SOD, localized primarily to the cytosol but also found in nuclei, lysosomes, and peroxisomes; SOD2 or MnSOD, localized in the mitochondria matrix; and SOD3, located in the extracellular matrix. SOD catalyzes a stepwise oxidation and reduction reactions, termed as dismutation, for affecting the conversion of O₂ ^.− to H₂O₂ [extensively reviewed in (99)]. Subsequently, H₂O₂ is converted to H₂O and O₂ by the antioxidant catalase, which is primarily localized to the peroxisome and the cytosol (126, 290). Another major antioxidant enzyme involved in the detoxification of H₂O₂ is GPx (277). GPx reduces H₂O₂ by the oxidation of GSH to produce oxidized GSH and H₂O. Oxidized GSH is then recycled to form reduced GSH by GR (267). The Trx-dependent Prx is also a major player in the antioxidant defense system. Prx is found in the nucleus, mitochondria, ER, peroxisome and the cytosol, depending on its various isoforms. The antioxidant protein functions to reduce peroxides, H₂O₂, and peroxynitrite (ONOO⁻) by using its thiol groups. For example, its thiol groups are oxidized to form disulfides, which are subsequently reduced by Trx to regenerate a functional Prx (121). These antioxidants function to ensure that a homeostatic redox milieu is in place to prevent excessive accumulation of ROS that could carry detrimental effects to the cell. As such, alterations in the cellular antioxidant defense systems, in addition to the aberrant generation of ROS, contribute significantly to the generation of the pro-oxidant intracellular environment that is associated with various processes of tumorigenesis. For example, this has been elegantly demonstrated by studies linking the varied expression of SOD2 to the different stages of tumorigenesis and its progression (73, 177, 203). Similar effects of Prxs have been documented and elegantly reviewed in Refs (116, 239). And perhaps the most commonly observed differences between transformed cells and noncancerous cells relate to the GSH metabolism with profound effects on cellular redox metabolism and cell fate (39, 82). These are just a few examples to highlight the importance that the balance between cellular ROS generation and antioxidant defense systems plays in altering cellular homeostasis and promoting abnormal cell growth and/or proliferation, thereby endowing cells with two critical hallmarks of cancer, that is, increased proliferation and death resistance (119).

The dichotomy of redox signaling in cell fate decisions

Depending on the concentration, ROS can evoke a myriad of downstream events ranging from cell proliferation and survival to cell death (Fig. 2) (13, 14, 155, 188, 240, 246, 254, 330, 332). Although sublethal levels of ROS have been shown to increase proliferation and enhance cell survival through the modulation of various phosphatase and protein kinases (14, 90, 136, 150, 197), an overwhelming increase in ROS is associated with macromolecular damage, such as lipid peroxidation, protein oxidation and inactivation, and DNA damage (115, 188, 197, 293, 294). The latter overwhelming increase in ROS, in the presence of intact tumor suppressor p53, further triggers apoptotic cell death (210), thus facilitating the removal of cells with irreparable lesions. Apart from the induction of p53-mediated apoptosis, ROS could also induce cell death via the sustained activation of c-Jun N-terminal kinase (JNK). The latter involves ROS-dependent inactivation of the mitogen-activated protein kinase (MAPK) phosphatase (MKP) that regulates the phosphorylation/activation of JNK by catalytic oxidation of cysteine to sulfenic acid (155). In a separate study, ROS-induced activation of apoptosis signal-regulating kinase-1 (ASK1) has also been implicated in the downstream activation of JNK/p38 MAPK (246, 330). In addition, a recent study further provides support to ROS-induced cell death via the disruption of iron metabolism, in which the use of pharmacological ascorbate was capable of increasing redox active labile iron for ascorbate toxicity via the H₂O₂-mediated disruption of iron–sulfur clusters (287). These results serve to reinforce the notion that ROS-induced changes in cellular kinase(s) and/or protein phosphatase(s) are critical determinants of a favorable response to death stimuli.

OPEN IN VIEWER

FIG. 2.

The biphasic effects of ROS. The potential biphasic outcomes of ROS depending on its different levels in different cell types, duration, space, and antioxidant defense system, that is, moderate ROS level induces cell survival and proliferation whereas high ROS level induces oxidative stress, DNA damage, and cell death. The biphasic effects of ROS could potentially regulate proteins/oncoproteins/tumor suppressor such as Bcl-2 (The antiapoptotic protein Bcl-2 section), STAT3 (STAT3 promotes survival and regulates ROS in a noncanonical manner section), PI3K and phosphatase when ROS is upregulated at levels that induce cell survival and proliferation as well as ASK, JNK, and MKP and p53 when ROS is upregulated at levels that induce detrimental effects; that is, cell death. ASK, apoptosis signal-regulating kinase; Bcl-2, B-cell lymphoma 2; JNK, c-Jun N-terminal kinase; MKP, mitogen-activated protein kinase phosphatase; PI3K, phosphoinositide 3-kinase; STAT, signal transducer and activator of transcription.

The dogmatic view of ROS from the standpoint of cell fate determination has undergone a radical change over the past couple of decades, thanks to a number of convincing reports providing clear evidence that ROS can also function as second messengers at concentrations that do not overwhelm the cells' antioxidant defense systems (13, 14, 188, 197, 216). Interestingly, the prosurvival effects of ROS, much in common with the prodeath functions, also involve activation of downstream signaling via oxidation of thiol groups of kinases or phosphatases. However, such modifications are often reversible, thereby showcasing their deliberate purpose in cell signaling (188, 216). For example, H₂O₂ could oxidize cysteine residues within the tumor suppressor phosphatase and tensin homolog (PTEN), thereby resulting in the inactivation of PTEN and consequential activation of the phosphoinositide 3-kinase (PI3K) signaling pathway. This oxidative modification could be reversed by Trx-mediated reduction of PTEN, thereby resulting in the restoration of PTEN phosphatase activity (188). Corroborating that, another study showed that elevated O₂ ^.− could similarly induce a reversible oxidization of PTEN, which leads to an increased phosphatidylinositol (3,4,5)-trisphosphate and phosphorylation of protein kinase B (AKT), a mechanism similarly observed upon growth factor induction (197). Further evidence by another group also demonstrated that H₂O₂ could reversibly induce oxidation and inhibition of SH2-containing protein tyrosine phosphatases, which is crucial for mitogenic signaling by growth factor such as platelet-derived growth factor (PDGF) (216). These studies, therefore, support the role of ROS in signaling transduction independent of apoptotic pathways.

The strong evidence depicting the biphasic signaling of ROS, namely growth signaling or oxidative damage/cell death (155, 188, 210, 216, 254), therefore, begs the question: does there exist a fixed threshold of ROS across all cell types that could distinctly differentiate the prosurvival from the death-inducing activity of ROS? The presence of a uniformly fixed ROS levels across different cell types is highly unlikely and implausible, due to the differences in the ROS generating and detoxifying systems in different tissues of origin as well as the environmental factors that impact cellular redox state(s). It is more likely that each cell type possesses a distinct threshold, which could potentially be governed by multiple factors such as the spatial proximity of ROS and its target(s) and the production and turnover rates of ROS. Therefore, it is imperative to unravel the crosstalk between intracellular redox status, genetic and epigenetic factors, their products (oncoproteins), and environmental cues that are involved in the acquisition and progression of the cancer phenotype to be able to identify specific targets for intervention.

The antiapoptotic protein Bcl-2

The Bcl-2 family consists of proteins that have a significant impact on cell death and apoptosis. They can be categorized into two groups, namely the antiapoptotic and proapoptotic groups. The antiapoptotic proteins comprise Bcl-2, B-cell lymphoma extra large (Bcl xL), and induced myeloid leukemia cell differentiation proten (Mcl-1), whereas the proapoptotic proteins comprise Bcl-2 associated X protein (BAX), Bcl-2 homologous antagonist killer (BAK), p53 upregulated modulator of apoptosis (PUMA), phorbol-12-myristate-13-acetate-induced protein 1 (NOXA), Bcl-2 associated death promoter (BAD), BH3 interacting domain death antagonist (BID), Bcl-2-like protein 11 (BIM). The canonical activity of the antiapoptotic members of the Bcl-2 family is associated with their ability to bind and sequester the proapoptotic family members, thereby inhibiting their recruitment to the mitochondria and preventing mitochondria outer membrane permeabilization (MOMP) and the release of apoptosis amplification factors (i.e., cytochrome c) from the mitochondria (19, 36, 63, 76, 104, 153, 225, 232, 328, 355, 359). For example, study has reported that antiapoptotic Bcl-2, Bcl-xL, and Mcl-1 could sequester proapoptotic truncated-BID, BIM, or PUMA (161). Similarly, others have shown that Bcl-2 and Bcl-xL/Mcl-1 could inhibit BAX and BAK, respectively, which prevents MOMP and cytochrome c release (76, 359). In contrast, the proapoptotic PUMA and NOXA could sequester Bcl-xL and Mcl-1, respectively, thereby releasing BAX and BAK to induce apoptosis (161, 218). By dint of their opposing functions, this suggests that a balance between the pro- and antiapoptotic proteins dictates cellular response to death stimuli, such as chemotherapy-induced cell death that is dependent on MOMP (291). Since the discovery of the prototype antiapoptotic protein Bcl-2, which is frequently overexpressed in hematopoietic cancers such as follicular lymphoma t(14;18)(+), acute lymphoblastic leukemia, chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma, and diffuse large B-cell lymphoma (103, 131, 215, 226), a number of related proteins with similar functions have been identified, such as Bcl-xL and Mcl-1 (19, 63, 104, 341, 359). Overexpression of Bcl-2 or other functionally similar family members tilts the balance between the anti- and proapoptotic proteins in favor of the former, which in the clinical setting is associated with poor disease prognosis, tumor progression, and/or aggressive disease (103, 131, 367, 392).

Although the overexpression of Bcl-2 appears to play a critical role in the acquisition of the chemoresistant phenotype (46, 85), there is accumulating evidence to indicate that post-translational modification of Bcl-2 profoundly impacts its antiapoptotic activity. To that end, Bcl-2 phosphorylation was shown to increase its binding to Bax and Bak as well as reduce drug-induced cell death (49, 70). Other studies have also shown that increased Bcl-2 phosphorylation is capable of conferring resistance to chemotherapeutic drugs such as etoposide, doxorubicin, and paclitaxel (68, 206).

Intriguingly, a number of reports attributed the death-regulating activities of the pro- and antiapoptotic members (i.e., Bcl-2, Bcl-xL, and Bax) to the modulation of cellular redox metabolism (3, 46, 162, 165). The first observations linking Bcl-2 to cellular redox environment came from the report suggesting an antioxidant activity of Bcl-2 by virtue of its ability to regulate cellular antioxidant pathway(s) as well as induce resistance to ROS-induced cell death (135). Specifically, Bcl-2 was shown to protect cells from H₂O₂- and menadione-induced oxidative cell death as well as dexamethasone-induced lipid peroxidation (135). A separate study showed that Bcl-2 overexpression transcriptionally upregulated the mitochondrial antioxidant, SOD2 (163). These observations suggested two possible scenarios, first that Bcl-2 itself harbored antioxidant activity or second that the induction of antioxidant systems, such as SOD2, could be a feedback activation or adaptive response triggered by the pro-oxidant milieu supported by Bcl-2. Although there was scant evidence to support the former, work from Steinman provided support for the latter by demonstrating a significantly higher (up to 73% increase) SOD activity in a murine B-cell line overexpressing Bcl-2 in the absence of an exogenous stimulus, indicating an endogenously higher O₂ ^.− scavenging rate. In a different bacterial cell system, Steinman also showed that Bcl-2 overexpression increased the mutation rate (up to threefold) as well as increased transcription of KatG catalase–peroxidase (enzyme with catalase and peroxidase activities) (311). These findings provided support to the hypothesis that the increase in cellular antioxidant capacity upon overexpression of Bcl-2 was not a direct effect of Bcl-2 as an antioxidant, but rather a feedback response to the pro-oxidant milieu afforded by Bcl-2 overexpression. The latter was corroborated by findings in human leukemia cells that provided evidence that overexpression of Bcl-2 inhibited death receptor- and drug-induced apoptosis by inhibiting upstream caspase 8 activation through the intermediary involvement of intracellular O₂ ^.− (46). Furthermore, pharmacological inhibition (diphenyliodonium: DPI) of NOX, an O₂ ^.− producer, or transient transfection with the dominant negative form of Rac1 (Rac1 ^N17) that blocks NOX activity restored apoptosis sensitivity of Bcl-2 overexpressing cells. Conversely, the apoptosis sensitizing effect of Rac1 ^N17 in Bcl-2 overexpressing leukemia cells was blocked upon an increase in O₂ ^.− upon pharmacological inhibition of SOD1 (diethyldithiocarbamate). These robust findings not only indicate that Bcl-2 overexpression could induce a pro-oxidant milieu but also demonstrate a redox-dependent antiapoptotic activity elicited by Bcl-2 (46). To that end, Chen and Pervaiz showed that Bcl-2 overexpression induced a pro-oxidant milieu (increased O₂ ^.− production) by engaging mitochondrial respiration; Bcl-2 overexpressing cells displayed higher mitochondrial cytochrome c oxidase (COX) enzymatic activity, oxygen consumption rate, and ATP synthesis (34, 35). This implies that Bcl-2 overexpression is capable of increasing the intrinsic electron transport across the ETC, and hence the probability of electrons leaking onto O₂ to generate O₂ ^.−. Furthermore, increased localization of the subunit Vα of COX (COX Vα), the rate-limiting enzyme of the ETC, to the mitochondria, as well as its interaction with Bcl-2, was demonstrated in cells overexpressing Bcl-2 (35). The link between Bcl-2 and a pro-oxidant tumor intracellular milieu was further reinforced by the discovery of a novel interaction between Bcl-2 and the small GTPase Rac1, which is involved in NOX assembly and O₂ ^.− production (342).

In light of the evidence linking Bcl-2 overexpression to an increase in intracellular O₂ ^.− and attributing a noncanonical antiapoptotic activity to the protein, how does one reconcile with the earlier findings that Bcl-2 inhibited oxidative stress-induced death signaling? One plausible explanation, presented in a preceding section, could be the feedback induction of antioxidant defense systems in cells overexpressing Bcl-2; however, a more convincing and direct evidence was provided by observations indicating a “redox rheostat” activity of Bcl-2 (Fig. 3d). Although Bcl-2 overexpression supported a pro-oxidant state under states of normoxia, this effect was remarkably reversed in the face of overt oxidative stress; Bcl-2 overexpression prevented further increase in mitochondrial O₂ ^.− production by decreasing COX activity and clamping down oxygen consumption in cells exposed to exogenous stimuli that induced oxidative stress such as antimycin A, hypoxia, serum withdrawal, and glucose deprivation (34, 35). From the standpoint of the functional biology of Bcl-2, the opposing redox modifying activities of the protein could be critical in promoting the process of tumorigenesis and its progression. A mild increase in ROS levels during states of normoxia could provide cancer cells with a survival advantage via a stimulatory effect on cell proliferation, migration, and invasion (198, 331), whereas in the setting of overt oxidative stress (such as induced upon exposure to DNA damaging agents and γ-irradiation (11, 85, 270, 293, 294)), the ability to clamp down on a further build up of ROS could provide a protective mechanism for cancer cells to evade chemotherapy-induced execution.

OPEN IN VIEWER

FIG. 3.

Oncoprotein signaling and ROS. (a) STAT3, STAT5, Rac1, and gelsolin act to increase ROS levels by multitude of mechanisms. (b) Under conditions of chemotherapeutic-induced oxidative stress, Bcl-xL overexpression and Mcl-1 overexpression prevent cell death possibly by decreasing ROS levels. (c) NRF2 regulates the antioxidant response, thereby affecting the intracellular redox milieu. NRF2 expression is potentially regulated differently at early and late stages of tumors. (d) Bcl-2 overexpression at physiological levels has been reported to enhance ROS production through increased mitochondrial localization of COX Vα, COX activity, and mitochondrial respiration and interaction with Rac1. Under oxidative stress conditions, however, Bcl-2 overexpression clamps down further increase in ROS levels by reducing COX activity and maintaining the mitochondrial respiration rate. (e) Myc potentially possesses dual redox function as it has been shown to regulate mitochondria biogenesis, CYP2C9 expression, and SOD2 expression to induce ROS production. However, under oxidative stress conditions, Myc could induce expression of γ-GCS as well as Prx5, thereby reducing ROS levels through an enhanced antioxidative capacity. (f) RAS also potentially possesses dual redox function. At physiological conditions, RAS has been shown to decrease expression of SOD2 and catalase, inhibit Sestrin 1/3, and activate Rac1 and NOX, thereby increasing ROS production. However, under oxidative stress, RAS can induce expression of antioxidant proteins through Pim-1 kinase as well as NRF2. Bcl-xL, B-cell lymphoma extra large; COX, cytochrome c oxidase; COX Vα, Vα subunit of COX; GRIM-19, gene associated with retinoid interferon-induced cell mortality 19; Mcl-1, induced myeloid leukemia cell differentiation protein; NRF2, NF-E2-related factor 2; SOD2, manganese superoxide dismutase.

Interestingly, there appears to be a reciprocity vis a vis the relationship between the Bcl-2 family and cellular redox status. Not only does overexpression of Bcl-2 impact intracellular redox status by regulating O₂ ^.− production under different metabolic states but also an increase in intracellular ROS has been linked to the post-translational modification of Bcl-2. In this regard, there is evidence to indicate direct oxidative modification of Bcl-2 (208), as well as our recent work underscoring the effect of an increase in intracellular O₂ ^.− on the sustained phosphorylation of Bcl-2 at serine 70 via nitrative modification and inactivation of the phosphatase protein phosphatase 2A (PP2A) subunit B56δ, responsible for Bcl-2 dephosphorylation. Notably, the redox-mediated sustained phosphorylation of Bcl-2 at serine 70 not only induced resistance to apoptosis in vitro, but was also associated with poor prognosis and higher disease grade in a small group of lymphoma patients (206). The phosphorylation of Bcl-2, in turn, could regulate intracellular ROS by influencing the expression of antioxidant enzymes, as demonstrated by Deng et al. (69). These findings collectively suggest distinct functional outcomes of Bcl-2 overexpression, whereby (i) under physiological states, Bcl-2 overexpression supports a pro-oxidant intracellular milieu, which influences its phosphorylation status by redox-dependent inactivation of PP2A (particularly at serine 70) and stabilizes its antiapoptotic activity and (ii) sustained serine 70 phosphorylation of Bcl-2 prevents the deleterious build up of ROS particularly during oxidative stress, thus blunting oxidative stress-induced execution that underlies the mechanism of anticancer activity of a number of clinically employed chemotherapeutic drugs and γ-irradiation. These observations lend credence to the hypothesis that Bcl-2 serves a “redox rheostat” function contingent upon the cellular redox environment and its phosphorylation status.

Death inhibitory function of Mcl-1 or Bcl-xL is associated with redox modulatory activity

While discussing the antiapoptotic activity of the Bcl-2 family, two other members deserve special mention, namely Mcl-1 and Bcl-xL. Overexpression of Mcl-1 or Bcl-xL, similar to Bcl-2, is a frequent finding in aggressive and chemoresistant cancers, and associated with poor disease prognosis/outcome. Mcl-1 is overexpressed in hepatocellular carcinoma, multiple myeloma, high-grade B-cell lymphomas, and plasma cell myeloma, and its expression in multiple myeloma is linked to disease relapse and shorter life span (42, 299, 367). In contrast, Bcl-xL is overexpressed in colorectal and tongue carcinoma, in which its expression is also positively correlated with malignant tumor staging as well as metastasis in tongue carcinoma (284, 392). Interestingly, in addition to antagonizing the proapoptotic proteins of the Bcl-2 family (19, 41, 63, 104, 302, 341, 359, 391), Mcl-1 and Bcl-xL have also been implicated in the regulation of ROS (Fig. 3b). For example, Mcl-1 was shown to suppress doxorubicin-induced increase in intracellular ROS production (H₂O₂ and mitochondrial O₂ ^.−) in p53-negative HCT116 colon carcinoma cells, which could be alleviated by gene knockdown of Mcl-1 (22, 64). The same research group further showed that knockdown of Mcl-1 increased NOX4 expression and NOX4 mitochondrial localization upon doxorubicin treatment, indicating that trafficking of NOX4 to the mitochondria may be crucial for drug-induced increase in ROS production in the absence of Mcl-1 (64). These findings provide testimony to the ROS regulating activity of Mcl-1 via mechanisms that suppress NOX4 expression, its mitochondrial localization, and ultimately mitochondrial ROS generation. Mcl-1 protein expression was also shown to be affected upon hypoxia-induced cell death. It was reported that hypoxia could reduce Mcl-1 level (33). As hypoxia induces ROS production such as mitochondrial O₂ ^.− (34), it could be hypothesized that hypoxia-induced reduction of Mcl-1 is a mechanism that contributes to the rise in ROS level and subsequent cell death. With evidence showing that Mcl-1 overexpression could reduce hypoxia-induced cell death, it is likely that the antioxidant role of Mcl-1 could play a part in reducing ROS, thereby protecting cancers cell from ROS-dependent hypoxia-induced cell death. Similarly, overexpression of the antiapoptotic protein Bcl-xL significantly inhibited intracellular ROS in response to a variety of ROS-inducing triggers such as tumor necrosis factor alpha (TNF-α), serum, or glucose deprivation and exogenous H₂O₂ (106, 162, 189, 251). Although the precise mechanism underlying the redox regulatory activity of Bcl-xL remains undefined, Bcl-xL does not behave as an antioxidant, but instead maintains mitochondrial physiology, thereby controlling mitochondrial ROS production (106). Supporting the latter is the finding that TNF-α-induced ROS and subsequent cell death require the protein ROS modulator 1 (Romo-1). Romo-1 was shown to decrease mitochondrial membrane potential and subsequently induce ROS production (CM-H₂DCFDA [2′7′-dichlorofluorescin diacetate]) through its inhibitory interaction with Bcl-xL. Importantly, overexpression of Bcl-xL could reverse TNF-α-induced ROS and cell death (162). These observations suggest a possible involvement of Bcl-xL in maintaining mitochondrial homeostasis by preventing excessive generation of stimulus-induced ROS, and in doing so inhibiting death execution.

Targeting the antioxidant side of Bcl-2 as a novel strategy to overcome chemotherapy resistance

There appears to be some degree of overlap in the noncanonical redox modulating activity of the major antiapoptotic proteins of the Bcl-2 family, which presents as a natural target for the development and design of therapeutic strategies against cancers rendered refractory by the aberrant expression of these prosurvival proteins. This acquires even more relevance given that a number of chemotherapy agents induce execution of cancer cells via mechanism that potentially involve a significant increase in intracellular ROS (270, 293, 294). It is plausible that the aberrant expression of proteins such as Bcl-2, Mcl-1, or Bcl-xL promotes drug resistance by impeding chemotherapy-induced oxidative stress. Therefore, it is highly desirable to identify novel therapeutic strategies and drugs that directly and/or indirectly inhibit the expression, phosphorylation (specifically serine 70), and activity of Bcl-2. To that end, the heightened interest and focus on the development of novel Bcl-2 inhibitors are a promising step in the right direction with potential therapeutic implications. Multiple Bcl-2 homology (BH) mimetics such as ABT737, ABT263, and ABT199 have been used empirically as well as in clinical trials to target cancers such as lymphoma, myeloma, and leukemia (1, 32, 56, 132, 146, 160, 167, 171, 252, 258, 276, 307, 317, 339, 344, 361). Although the success of some of these trials was compromised by the nonspecific targeting of Bcl-xL and the development of severe thrombocytopenia, the development of a more selective inhibitor, ABT199 (venetoclax), which does not target Bcl-xL and spares platelets, is extremely encouraging and has received FDA approval as a monotherapy for CLL (62, 307, 339, 344). Interestingly, a recent study linked the Bcl-2 inhibitory activity of ABT199 to an increase in ROS production (Carboxy-H₂DCFDA), which further supports the redox regulatory role of Bcl-2 in cancer cells (376). Given these data, it is tempting to speculate that the increase in oxidative stress observed in cancer cells upon exposure to ABT199 could be a function of inhibiting the redox rheostat activity of Bcl-2 by preventing its phosphorylation at serine 70, thereby restoring apoptosis sensitivity of cancer cells rendered refractory to chemotherapy by the sustained expression for Bcl-2. Similarly, as alluded to the preceding section, given that Mcl-1 and Bcl-xL are upregulated in many human cancers and their ability to induce resistance to ROS-mediated execution (22, 42, 64, 106, 162, 189, 251, 284, 299, 367, 392), it is only logical to design and develop small molecules that specifically target the redox modulating activity of these proteins. Some examples of selective inhibitors for Mcl-1 include A-1210477 and S63845, whereas those for Bcl-xL include WEHI-539 and A-1155463 (175, 190, 191, 326). It remains to be seen whether the death sensitizing effects of these selective compounds also involve neutralizing the redox regulatory functions of these proteins, similar to what has been reported for the Bcl-2 inhibitor ABT199.

STAT3 promotes survival and regulates ROS in a noncanonical manner

The signal transducer and activator of transcription (STAT) proteins belong to a family of transcription factors involved in the regulation of several cellular processes, including apoptosis and cell cycle progression (123, 306, 375). Specifically, the STAT proteins are involved in the JAK-STAT pathway and activated by factors or ligands such as growth factors (e.g., epidermal growth factor [EGF] or PDGF) and cytokines (e.g., interleukin-6 [IL-6]). Upon ligand binding, the receptor subunits dimerize, thereby recruiting the Janus kinase (JAK) family proteins to proximity for their transphosphorylation and activation at the cytoplasmic domain. Upon activation, JAKs phosphorylate STAT proteins, which upon phosphorylation form hetero- or homodimers, translocate to the nucleus wherein they bind consensus DNA-recognition motifs, found in the promoter region of the target genes, thus activating gene transcription (54, 55, 142). One notable activator or ligand for one such family member, STAT3, is IL-6 (Fig. 4) (172), a cytokine associated with poor prognosis in breast cancer patients and implicated in the progression of lung carcinoma (282, 384).

OPEN IN VIEWER

FIG. 4.

Cellular redox status in STAT3-mediated oncogenic signaling. Phosphorylation of Y705 residues results in the shuttling of STAT3 homo- or heterodimers to the nucleus where it binds to consensus DNA-recognition motifs in the promoter region of its target genes and activate transcription. Among the STAT3 target genes, Bcl-2 and Bcl-xL are responsible for antiapoptotic signaling, whereas MMP-2 and MMP-9 are responsible for metastasis. Another target, NOX4, is proposed to promote IL-6 production through ROS production, thus enhancing STAT3 activation through a positive feedback loop. Binding of STAT3 to GRIM-19 shuttles the complex to the ETC at complex I, thereby potentially affecting ROS production at the mitochondria. Phosphorylated STAT3 has also been shown to interplay with phosphorylated Bcl-2 via ROS. IL-6, interleukin-6; MMP, matrix metalloproteinase.

STAT3 has been most frequently associated with tumorigenesis and is widely documented as an oncoprotein. Aberrant STAT3 signaling, or the upregulation of STAT3 phosphorylation at tyrosine 705 (Y705), positively associated with JAK expression or human papillomavirus infection, is observed in a variety of cancers such as breast, lung, and cervix (139, 204, 298, 375). As a transcription factor, STAT3 induces the transcription of many target genes, most of which are crucial for growth, survival, cell transformation, angiogenesis, and metastasis in tumorigenesis (Fig. 4). One study showed that immortalized human breast epithelial cells could undergo cell transformation when a constitutively active STAT3 mutant (STAT3-C) is expressed. This was further shown to be dependent on the ability of STAT3-C to upregulate matrix metalloproteinase (MMP)-9 messenger ribonucleic acid (mRNA) and protein levels in these immortalized breast epithelial cells (61). Another group has shown that increased MMP-2 expression is also correlated with increased STAT3 activity in highly metastatic melanoma cells. They further demonstrated that transfection of STAT3-C and dominant negative STAT3 (STAT3-DN) reciprocally activated and inhibited MMP-2 promoter, respectively. In addition, the transfection of STAT3-DN in metastatic cells decreased the expression of MMP-2. Importantly, in vivo study through the injection of metastatic cells harboring STAT3-DN in mice also demonstrated a decrease in tumor cell growth and metastasis (372). In addition, STAT3 contributes to angiogenesis through the induction of vascular endothelial growth factor (VEGF), a key mediator of angiogenesis in cancer. The upregulation of VEGF was observed, concomitant with increased STAT3 activity in breast and head and neck cancers. This association was further corroborated when STAT3-C was stably expressed in murine NIH3T3 fibroblast and melanoma B16 cells. Importantly, the increased vascularization, as confirmed by higher hemoglobin content, was observed when STAT3-C-transfected B16 tumor cells were implanted in vivo (245). Corroborating that, another study also showed that targeting STAT3 with a small-molecule inhibitor could block VEGF expression in vitro as well as inhibit tumor growth and angiogenesis in vivo (245, 377). Apart from angiogenesis, constitutive STAT3 activation has been widely described to inhibit apoptosis through activation of antiapoptotic genes such as Bcl-2 and Bcl-xL (31, 98). Inhibition of STAT3 reduces expression of the antiapoptotic proteins and induces apoptosis (8, 84, 241). Intriguingly, Choi et al. reported the use of novel inhibitor of STAT3, tuberatolide B (TTB) to induce apoptotic cell death. TTB selectively inhibits STAT3 phosphorylation and transcriptional activity, reduces downstream targets of STAT3 as well as induces apoptotic cell death. Of note, the ROS scavenger, N-acetyl-L-cysteine (NAC), blocked the effects of TTB on ROS production (H₂DCFDA), STAT3, and its target genes as well as cell death, indicating that the effect of TTB is dependent on ROS (45). Although a causal relationship between TTB-STAT3 apoptosis was not investigated here, these findings nonetheless suggest a role of ROS in regulating STAT3 activity. In this regard, several studies have demonstrated the effects of ROS on STAT3 as illustrated in Figure 3a. NOX has been reported to indirectly activate STAT3 by stimulating IL-6 production and subsequent activation of the JAK1-STAT3 pathway. Overexpression of NOX4 in nonsmall cell lung cancer (NSCLC) cells resulted in ROS-dependent IL-6 production as well as STAT3 phosphorylation at Y705, whereas NOX4 knockdown subsequently reduced ROS production (H₂O₂) and suppressed IL-6 production and STAT3 activity. Interestingly, the exogenous introduction of IL-6 and subsequent STAT3 activation could also upregulate NOX4. This collectively suggests a positive loop whereby increased IL-6 production and IL-6/STAT3 activation could increase NOX4 expression, which, in turn, upregulates ROS production to further enhance IL-6 production and IL-6/STAT3 activation (194). These data are further corroborated by a study that linked pharmacological inhibition of NOX (and downstream ROS production) (206) to a significant decrease in STAT3 activation in hepatocellular carcinoma cells (308). Corroborating that, our group demonstrated that exposure of cells to the NOX inhibitor, DPI, could reverse the induction of STAT3 phosphorylation in Bcl-2 overexpressing CEM lymphoblastic leukemia cells, which constitutively harbor elevated O₂ ^.− levels (34, 156).

Although ROS have been linked to the activation of STAT3, STAT3 activation, in turn, could also regulate intracellular ROS production. In this case, a noncanonical role of STAT3, independent of its transcriptional activity, was implicated in the regulation of mitochondrial O₂ ^.− production. Overexpression of wild-type STAT3 resulted in an increase in mitochondrial O₂ ^.− production, which was reversed by a functional STAT3 mutant (STAT3-Y705F). Another group also demonstrated that gene associated with retinoid interferon-induced cell mortality 19 (GRIM-19) is capable of acting as a chaperone to recruit STAT3 to the mitochondria (318). This suggests that STAT3 could be inducing mitochondrial O₂ ^.− via its localization to the mitochondria (Fig. 4). In support of this, STAT3-deficient cells (STAT3^−/−) exhibit decreases in mitochondrial complexes I and II activities and reconstitution of STAT3 into STAT3^−/− cells rescued the loss of ETC activity, thereby supporting the role of STAT3 in the regulation of mitochondrial redox metabolism (354). In addition, we previously reported a crosstalk between increased expression of the antiapoptotic protein, Bcl-2, cellular pro-oxidant milieu, and phosphorylation/activation of STAT3 (pY705); Bcl-2 overexpression induced an increase in STAT3 phosphorylation at Y705, which was reversed by DPI and Rac1 inhibitor. Of note, using site-directed mutagenesis (Y705F), STAT3 activation was associated with an increase in Bcl-2 phosphorylation at serine 70, which stabilized its antiapoptotic activity (34, 46, 156, 206, 342). Collectively, these data point to the existence of a positive feed forward loop between Bcl-2 and STAT3 phosphorylation, whereby an increase in mitochondrial O₂ ^.− upon Bcl-2 overexpression induces STAT3 phosphorylation/activation, which, in turn, further augments ROS levels to promote serine 70 phosphorylation (and activation) of Bcl-2. These findings provide testimony that not only does cellular redox status positively impact STAT3 activation but also that the activated STAT3 amplifies this circuit by further fueling mitochondrial ROS generation (Fig. 4) (156).

Targeting the redox-modifying effect of STAT3 as an anticancer strategy

Based on the evidence linking STAT3 activation to a host of human cancers and its upregulation as a surrogate survival mechanism in cells rendered resistant to tyrosine kinase inhibitor (TKI), the transcription factor has been an attractive target for cancer therapy (139, 204, 298, 375). Intriguingly, STAT3 inhibitory effect was discovered as an off-target effect of some drugs developed for the treatment of unrelated indications, which led to the repurposing of these compounds for the treatment of cancers with elevated STAT3 expression (6, 235). One example is nifuroxazide, which has been in use since 1966 for the treatment of colitis and diarrhea. Notably, this antibiotic was shown to inhibit the phosphorylation of STAT3 at Y705 (236) and elicited antitumor and antimetastatic effects in colorectal cancer and melanoma cells (383, 398). Nifuroxazide reduced cell viability by triggering Bax-dependent apoptosis, as well as repressed two STAT3 target genes MMP-2 and MMP-9 that are involved in metastasis. It should be pointed out that the apoptosis-inducing activity of nifuroxazide was associated with an increase in intracellular ROS (mitochondrial O₂ ^.− and H₂O₂); however, the precise mechanism underlying the involvement of ROS in this context is not well understood. A plausible scenario could be an increase in ROS triggered by an increased shift to a ratio of Bax to Bcl-2 upon inhibiting STAT3 activation. In this regard, mitochondrial translocation of Bax has previously been shown to trigger an increase in intracellular ROS (CM-H₂DCFDA and dihydroethidium [DHE]) (3, 165).

Another STAT3 inhibitor, NSC-743380, has been shown to display antitumor effects against NSCLC cells by inhibiting STAT3 phosphorylation (Y705) (110) and inducing oxidative stress (201). Interestingly, the antioxidant, nordihydroguaiaretic acid, was able to reduce ROS level (H₂DCFDA) and rescue cells from the death-inducing effects of NSC-743380, without affecting its STAT3 inhibitory activity, thus indicating a downstream signaling role for ROS in therapies aimed at targeting STAT3 activation. Indeed, knockdown of STAT3 increases ROS levels and suppresses tumor growth. More recently, Wong et al. reported promising antitumor effects of a first-in-man direct STAT3 inhibitor, OPB-51602, in a Phase I clinical study involving refractory solid malignancies (363). The molecular mechanisms underlying the antitumor activity of OPB-51602 also involve a strong mitochondrial redox component (Hirpara et al., unpublished results), thus underscoring the crosstalk between STAT3 and cellular redox metabolism.

Nevertheless, how does one reconcile with the similar effects on ROS production upon activation or pharmacological inhibition of STAT3? In light of the data supporting the translocation of activated STAT3 to the mitochondria and its downstream effect on mitochondrial metabolism, such as O₂ ^.− production (156, 354), it is plausible that activated STAT3 amplifies mitochondrial ETC activity via its interaction with complex I component(s) (354), hence impacting oxygen consumption and as such increasing the probability of electrons leaking onto oxygen to generate O₂ ^.−. This is similar to what has previously been shown by our group upon overexpression of Bcl-2 (34, 35). This assumption is further supported by the observation linking increased STAT3 phosphorylation to enhanced mitochondrial oxidative phosphorylation (OXPHOS) in drug-resistant cancer cells [(187) and Hirpara et al. unpublished results], which creates a synthetic lethal condition that relies on OXPHOS. This allows the targeting of mitochondrial ETC to shut down the forward transport of electrons (similar to rotenone) and in doing so triggers oxidative stress. Alternatively, the redox effect of shutting down STAT3 signaling could also be a function of inhibition of two transcriptional targets of STAT3, Bcl-2, and Bcl-xL. Downregulation of Bcl-2 or Bcl-xL could tilt the balance in favor of Bax, which could drive mitochondrial ROS production (3, 31, 98, 165), as discussed previously.

The neglected STAT5 in tumorigenesis

In addition to STAT3, STAT5, referring to both STAT5a and STAT5b isoforms, has also displayed tumorigenic properties (368). Constitutive activation of both STAT3 and STAT5 has also been reported in breast cancer (346). With that in mind, STAT5 is postulated to function as an oncoprotein through regulation of several pathways. STAT5 activation has been described to inhibit apoptosis through its role as a transcription factor in regulating genes involved in antiapoptotic signaling, such as Bcl-2 (219). Knockdown of STAT5b and expression of dominant-negative STAT5b induced G1 arrest and subsequent cell death, highlighting the role of STAT5 in the inhibition of apoptosis (219). In addition, knockdown of STAT5 significantly reduced the expression of proteins involved in metastasis such as focal adhesion kinase, VEGF, and MMP-2 together with a reciprocal increase in E-cadherin. These findings underscore the involvement of STAT5 in the activation of cell invasion and metastasis (374).

One of the activators of STAT5 signaling pathway in acute myeloid leukemia (AML) is FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD). This strong association between STAT5 and FLT3-LM was observed in primary AML blast cells. In addition, introduction of the FLT3 mutants increased the transforming potential of interleukin-3-dependent murine pro-B cells (Ba/F3) in vitro (309). More relevantly, a separate study showed that FLT3-ITD expression in AML cells induced an increase in intracellular H₂O₂ in an FLT3- and NOX-dependent manner; PKC412, an inhibitor of FLT3, as well as NOX inhibitors DPI and VAS2870, significantly reduced ROS levels in FLT3-ITD-expressing AML cells. Importantly, knockdown of p22phox, a component of NOX, is capable of reducing STAT5 phosphorylation and signaling in FLT3-ITD-expressing AML cells, suggesting that FLT3-induced H₂O₂ via NOX/p22phox is crucial for STAT5 signaling (365). The latter is further supported by observations highlighting the critical involvement of the small GTPase Rac1 in the crosstalk between STAT5 and ROS; FLT3-ITD-induced ROS generation (H₂DCFDA) was shown to require both activated STAT5 and Rac1, as silencing of STAT5, inhibition of Rac1, or expression of Rac^N17 alleviated ROS levels. In addition, the same study also reported that phosphorylated STAT5 could bind to Rac1, suggesting that this binding may be a mechanism of FLT3-ITD/STAT5-mediated ROS production (281). Furthermore, the binding of Rac1 to phosphorylated STAT5, in this case STAT5a, was believed to be a requirement for its nuclear translocation, as presence of active Rac1 (Rac1 ^V12) and depletion of Rac1 reciprocally affect the nuclear localization of phosphorylated STAT5a (157). Given the function of Rac1 in ROS production, the binding of two proteins strongly argues in favor of an involvement of ROS in the nuclear translocation of STAT5. Moreover, STAT5 activation has also been associated with increased ROS production (Carboxy-H₂DCFDA) in FLT3-ITD-expressing AML cells via increased expression of its transcriptional target NOX4 (150). Of note, knockdown of NOX4 reduced ROS levels and attenuated FLT3-ITD-induced transformation. These findings point to yet again another positive loop between NOX and STAT5, whereby activated STAT5 is capable of increasing NOX expression, thereby increasing ROS level to activate STAT5, similar to STAT3 (Fig. 3a).

STAT5 is also one of the significant drivers of chronic myelogenous leukemia (CML) and was observed to positively correlate with BCR-ABL mutation rate. Intriguingly, STAT5 is proposed to enhance mutation rate of BCR-ABL via ROS production in CML cells, as shown by the increased ROS-dependent DNA damage. Overexpression of STAT5 was accompanied by an increase in ROS level (DHE) independent of JAK2 but dependent on BCR-ABL signaling. STAT5-induced ROS production subsequently resulted in DNA double strand breaks, indicated by increased γH2AX (phosphorylated histone H2AX) level, which could be rescued by NAC. As a result, the potential STAT5-induced ROS-mediated BCR-ABL mutation was proposed to reduce sensitivity to TKIs as a mechanism of TKI resistance in BCR-ABL-driven CML cells (352). These findings are corroborated by the significantly high STAT5 expression in patients with acquired resistance to BCR-ABL inhibitors, such as imatinib (353), thus suggesting the involvement of ROS in mediating STAT5-induced tumorigenesis.

Targeting STAT5-ROS nexus as an anticancer strategy

The crosstalk between STAT5 and intracellular ROS appears to play a critical role in STAT5-induced tumorigenesis and/or its progression, and as such offers a window of opportunity for the design and development of specific STAT5 inhibitors for targeting FLT3-ITD-expressing AML as well as restoring the sensitivity of BCR-ABL-driven CML cells to TKI. However, unlike the abundant availability of STAT3 inhibitors, few STAT5 inhibitors have been developed. Nevertheless, in a repurposing approach, the antipsychotic drug, pimozide, has demonstrated inhibitory effect on STAT5 phosphorylation, resulting in apoptosis of AML cells and reduced tumor burden in FLT3-driven AML mouse model. In addition, pimozide could enhance the reduction of STAT5 phosphorylation as well as cell viability when treated with PKC412 or sunitinib, both TKIs (237). Interestingly, pimozide inhibited binding of STAT5 to NOX4 promoter, leading to a significant decrease in NOX4 mRNA expression (150). Although there is lack of evidence to associate pimozide-mediated STAT5 inhibition to a change in cellular redox status, the observed decrease in NOX4 mRNA would be expected to decrease ROS levels after STAT5 inhibition. Despite the relatively modest advance in the design of STAT5 inhibitors, the redox modulating effects of STAT5 could be a potential novel strategy for the design and development of new pharmacological agents for the management of AML and CML.

The therapeutic stakes of efficiently targeting mutant RAS-driven cancers

Inhibition of oncogenic RAS has become an important therapeutic strategy against RAS-driven cancers. However, past attempts at drugging RAS have not had the desired results. Nevertheless, continuous effort in understanding the protein as well as its structure for therapeutic intervention has been on-going (207, 213, 360). The efficacy of these strategies remains to be fully evaluated and the rather disappointing results with farnesyl transferase inhibitors have left an open area for developing novel strategic therapeutics (47, 86, 207, 360, 378, 381, 399). As mentioned previously, the ROS dependency of RAS-driven tumors may well present with a window of opportunity, which could be exploited for therapeutic management (79). The initial attempts in this respect involved alleviating intracellular ROS to levels that can no longer promote cellular proliferation. Interestingly, administration of antioxidant such as vitamin E for KRAS-pancreatic cancers elicited antiproliferative effects (347). The NOX inhibitor, DPI, has also been shown to deplete ROS level to promote apoptotic cell death in KRAS-mutated pancreatic cancer cell line PANC-1 through NOX4 inhibition (223). Similarly, metformin-induced growth inhibition of PANC-1 and Mia-PaCa cells carrying mutant KRAS was a function of decreased ROS production through upregulation of SOD2 and downregulation of NOX2 and NOX4 proteins (38).

The second type of strategy tested was to disrupt ROS homeostasis to reach lethal levels of ROS in oncogenic RAS-driven cancer cells (79, 279). Such a strategy was validated by a screening of >50,000 compounds, which resulted in the discovery of a class of molecules against mutant KRAS-expressing cells (289). Initially developed as a muscle relaxant, the lead molecule of this massive screen, lanperisone, induced cell death by increasing intracellular ROS (DCFDA), which was dependent on iron metabolism and activation of MAPK pathway (289). This molecule has been described to induce ferroptosis, a novel form of cell death that relies on iron-mediated ROS production, MAPK pathway, and mitochondrial porins voltage-dependent anion channel 2/3 (373). Another chemical library screening aimed at identifying compounds targeting lung adenocarcinoma cells harboring epidermal growth factor receptor or KRAS mutations identified a potential candidate, lung cancer screen 1 (LCS-1, which reduced proliferation and induced apoptosis through a mechanism that also involves inhibition of MAPK and PI3K pathways (304). Interestingly, further investigations into the biological activity of LCS-1 identified SOD1 as a likely target (303). Indeed silencing of SOD1 reduced cell growth of LCS-1-sensitive cells while its overexpression increased resistance to LCS-1. In addition, treatment with LCS-1 significantly reduced SOD1 activity (303). Interestingly, the use of vitamin C was shown to induce massive ROS accumulation (DCFDA) to kill mutant KRAS colorectal cancers. The effect was due to an increased uptake of oxidized vitamin C, termed dehydroascorbate (DHA), leading to oxidative stress via DHA depletion of GSH. Accumulated ROS subsequently inactivates glyceraldehyde 3-phosphate dehydrogenase, which in a highly glycolytic environment of KRAS mutant cells, leading to energetic crisis and cell death (388). Lastly, our group recently devised a novel strategy using a small compound with the ability to hyperactivate mutant KRAS harboring cells (as well as cells displaying HRAS and NRAS mutations), which resulted in AKT-dependent increase in mitochondrial O₂ ^.− and intracellular H₂O₂ production and cell death (147). In addition, cell death induced by this small molecule displayed features of both autophagy and apoptosis mediated by activation of ERK and JNK (364). Taken together, these results show that ROS production might be the Achilles heel of RAS-driven cancers and could be exploited for the specific targeting of cancers carrying RAS mutations.

ROS-dependent approaches to targeting the tumorigenic activity of Rac1

Given the fact that overexpression or gain of function mutations enhance the redox effects of Rac1, there have been attempts to employ ROS scavengers as an indirect means to block the oncogenic effects of Rac1 (209, 255). One notable example is the use of NAC to inhibit Kaposi's sarcoma (KS)-like tumor development in constitutively active Rac1 (RacCa) transgenic mice. This study showed that developed tumors of RacCa mice possess significantly higher ROS (O₂ ^.−) levels than those of wild-type mice. Although no direct measurement of ROS was employed after NAC treatment, RacCa mice supplemented with NAC did not develop any detectable tumor compared with their controls. In addition, supplementation of NAC after tumor onset was capable of deterring further tumor development. These observations, therefore, strongly suggest that ROS produced by constitutively active Rac1 fuel KS-like tumorigenesis and that antioxidants such as NAC could have potential chemopreventive value against ROS-induced tumorigenesis (209). Another notable mention is the Rac1 inhibitor, NSC-23766, which was shown to be effective in abrogating Rac1-induced resistance against cancer therapy. In a study involving breast cancer cells, the surviving fraction of cells after hyperfractionated radiation (HFR) was shown to harbor upregulated Rac1 expression and significant increases in the level/activation of prosurvival proteins, ERK1/2, NF-κB, Mcl-1, and Bcl-xL. A priori treatment with NSC-23766 resulted in a significant downregulation of prosurvival factors as well as inhibition in tumor colony forming ability after HFR (125). Although no ROS measurement was employed in this study, others have undoubtedly demonstrated that Rac1 inhibition with NSC-237766 could abrogate Rac1-induced ROS production (20, 83). These findings provide testimony that inhibition of Rac1 or its redox function could potentially shut down Rac1-dependent oncogenic effects.

On the flip side, studies have also taken the advantage of Rac1-induced ROS to induce apoptosis as a means to prevent tumor progression (164, 243). To that end, Rac1/NOX-dependent ROS production (DCFHDA) was shown to be the underlying mechanism of glutamate-induced execution of neuroblastoma cells. The authors demonstrated that glutamate-induced apoptosis was rescued upon treatment with either NAC, DPI, neopterine, or apocynin or upon transfection with the dominant negative Rac1^N17, thus underscoring the involvement of ROS in glutamate-induced apoptosis of neuroblastoma cells (243). Similarly, knockdown of NOX1 or transfection of Rac1^N17 not only reduced TNF-α-induced O₂ ^.− but also blocked Rac1/NOX1-dependent necrotic cell death (164). Furthermore, our group also provided evidence, although indirect, that simvastatin, a 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitor, -induced apoptosis involved non-canonical Rac1-induced O₂ ^.− signaling that triggered downstream JNK activation and JNK-induced pro-apoptotic BIM extra long expression in HCT166 cells (397). Taken together, these data highlight the importance of Rac1-induced ROS production in the process of tumorigenesis and its progression as well as a potential target for therapeutic exploitation. Although there is conflicting experimental evidence vis a vis the involvement of Rac1-driven ROS production in promoting or inhibiting tumorigenesis, the contradiction could be explained due in part to the different cellular context, tumor microenvironment, the expression pattern of ROS producers, and detoxifier (antioxidant) and constitutive redox metabolism, which governs the ROS threshold of different cancer cell types in determining cell survival or execution.

Drugging Myc: ROS dependent?

Due to the participation of Myc in tumorigenesis, it is again crucial to identify inhibitors or drugs that could shut down the oncogenic functions of Myc. A thorough review of Myc inhibitors was recently published by Whitfield et al., of which some are effective in vitro or in vivo as well as tested clinically (357). The compiled list comprises compounds that target Myc directly, via the interference of its transcriptional function, binding partner capability, and its expression, and indirectly, via the inference of its regulatory proteins and effect on Myc-driven tumors (357). Of these compounds and inhibitors, the compound 10058-F4 could relevantly and simultaneously affect redox status during its effect of decreasing ATP production and cell proliferation, arresting cell cycle and inducing apoptosis in ovarian cancer cells (348). A compound identified to inhibit the interaction of Myc and Max (385), 10058-F4, could significantly reduce intracellular and mitochondrial ROS levels (348). Although just an association between ROS reduction and apoptosis upon 10058-F4 treatment, this suggests that ROS is required for Myc-driven tumorigenesis as well as the manipulation of ROS level may be a major key to Myc-driven tumor execution.

The conundrum: Inhibit or activate NRF2 as a therapeutic strategy?

The seemingly disparate roles of NRF2 at different stages of the tumorigenesis process present biologists and clinicians with the obvious quandary of identifying specific instances wherein inhibiting NRF2 could be a favorable anticancer strategy as opposed to other scenarios wherein induced activity could lend therapeutic relevance. The answer would plausibly be dependent on cell type, ROS level, and the stages of cancer. On the one hand, inducers of NRF2 may prove to be useful at early stages of tumorigenesis as ROS could potentially be a driving force for cell transformation and initiation of cancer. Indeed, multiple reports have suggested that activating the Keap1-NRF2 pathway is a viable option for cancer preventive therapy. For example, sulforaphane, an activator of NRF2 that was isolated from broccoli, was reported to have anticarcinogenic effect (296, 394). Another potent NRF2 inducer, oltipraz, was also shown to exert chemopreventive effect against urinary bladder carcinogenesis through activation of NRF2 (143).

On the other hand, NRF2 has also been shown to be upregulated in head and neck cancer and play a role in chemoresistance, as well as its transcriptional activity has been positively correlated with the different stages of lung cancer (310, 325, 350). However, in comparison with the inducers of NRF2, the identification of small molecule inhibitors of NRF2 has not been overly successful. Nevertheless, as a repurposing strategy, dimethyl fumarate, approved for the treatment of multiple sclerosis and psoriasis, was reported to elicit strong antitumorigenic effect in vitro and in vivo at high dose by triggering oxidative stress via the reduction of protein deglycase (DJ-1)-mediated stabilization of NRF2 (280). Two other compounds, brusatol and luteolin, have also been reported to sensitize cancer cells to chemotherapeutic drugs, such as cisplatin, carboplatin, 5-fluorouracil, paclitaxel, etoposide, oxaliplatin, bleomycin, and doxorubicin, by reducing NRF2 protein level and its responses (275, 323). In addition, premalignant breast epithelial cells and breast cancer cells harboring loss/mutant BRCA1 were more susceptible to ROS accumulation and cisplatin-induced cytotoxicity due to the impaired NRF2-regulated antioxidant response (105). As several commonly used chemotherapeutic drugs trigger intracellular ROS generation/accumulation (270, 293, 294), the inhibition of NRF2 could possibly release the breaks on ROS-dependent execution (Fig. 6), thereby sensitizing cancer cells to chemotherapy as well as overcoming resistance to ROS-mediated therapies.

OPEN IN VIEWER

FIG. 6.

Targeting NRF2 as a means of chemotherapy via its activation or inhibition. (a) Inducers of NRF2, sulforaphane, and oltipraz can potentially act to reduce ROS levels required for ROS-induced tumor progression. (b) Inhibitors of NRF2, brusatol, luteolin, and high dose of DMF could reduce NRF2 expression levels, thereby potentially resulting in excessive ROS accumulation to induce cell death, particularly when employed with ROS-dependent chemotherapeutic drugs. DMF, dimethyl fumarate.