Redox Dysregulation in the Tumor Microenvironment Contributes to Cancer Metastasis

Abstract

Significance:

Redox dysregulation under pathological conditions results in excessive reactive oxygen species (ROS) accumulation, leading to oxidative stress and cellular oxidative damage. ROS function as a double-edged sword to modulate various types of cancer development and survival.

Recent Advances:

Emerging evidence has underlined that ROS impact the behavior of both cancer cells and tumor-associated stromal cells in the tumor microenvironment (TME), and these cells have developed complex systems to adapt to high ROS environments during cancer progression.

Critical Issues:

In this review, we integrated current progress regarding the impact of ROS on cancer cells and tumor-associated stromal cells in the TME and summarized how ROS production influences cancer cell behaviors. Then, we summarized the distinct effects of ROS during different stages of tumor metastasis. Finally, we discussed potential therapeutic strategies for modulating ROS for the treatment of cancer metastasis.

Future Directions:

Targeting the ROS regulation during cancer metastasis will provide important insights into the design of effective single or combinatorial cancer therapeutic strategies. Well-designed preclinical studies and clinical trials are urgently needed to understand the complex regulatory systems of ROS in the TME. Antioxid. Redox Signal. 39, 472–490.

Introduction

Reactive oxygen species (ROS) constitute a class of highly reactive oxygen-containing molecules that have been implicated in signal transduction pathways and control a range of crucial biological activities, such as cell quiescence, differentiation, and even apoptosis (Sies and Jones, 2020; Suhail et al., 2019). Dysregulated redox homeostasis is a common pathophysiological condition, and excessive ROS production causes oxidative stress, which leads to a variety of diseases, including cancer.

Metastasis is the last stage of cancer progression and is the main reason for antitumor therapy failure (Suhail et al., 2019). In fact, neoplastic cells alone are not sufficient for metastasis. With the “seed and soil” theory proposed by Stephen Paget in 1889 (Paget, 1989), the role of cancer stroma in promoting tumor metastasis has begun attracting increased attention in the field of cancer research in recent years. Metastasis is a multistep process in which metastasizing malignant cells undergo dramatic redox metabolic changes and exhibit high levels of oxidative stress; these cells thrive in diverse environments and then migrate and invade surrounding tissues (Vanharanta and Massague, 2013). The tumor microenvironment (TME) is a multicellular system that facilitates the successful survival and growth of rare cancer cells in “new soil” (Quail and Joyce, 2013). Hence, an imbalanced increase in ROS levels can change the biological state of tumor cells and stromal cells in the TME via complex cell signaling and the release of numerous regulatory factors, which ultimately contribute to tumor progression (Tasdogan et al., 2021).

This evidently close relationship between the TME and ROS prompted us to summarize the changes in ROS signaling during different metastatic steps and elucidate how tumor-associated stromal cells in the TME leverage ROS to promote cancer progression. Furthermore, we also summarized potential therapeutic strategies targeting ROS dysregulation with the aim of offering new opportunities to improve clinical applications.

ROS and Tumorigenesis

ROS generation

ROS is a collective term referring to highly active oxygen-containing molecules that are classified as free radical and nonradical forms. Free radical ROS include the superoxide anion (^•O₂ ⁻), the hydroxyl radical (^•OH), the peroxyl radical (ROO^•), and the alkoxyl radical (RO^•). Nonradical ROS include hydrogen peroxide (H₂O₂), singlet molecular oxygen (¹O₂), ozone (O₃), hypochlorous acid (HClO), and other similar compounds (Sies and Jones, 2020). The major endogenous sources of ROS are membrane-bound nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) and mitochondria (Fig. 1). NOXs constitute a group of transmembrane flavocytochrome proteins that transfer electrons from NADPH to flavin adenine dinucleotide (FAD) cofactors, catalyze the reaction to convert O₂ into ^•O₂ ⁻, and eventually form H₂O₂.

FIG. 1.

The major ROS and their subcellular sites of generation. ROS are mainly generated by membrane-bound NOXs and in mitochondria. NOXs widely distributed in biological membranes produce ROS via the catalysis of O₂ into ^•O₂ ⁻. SODs promote the dismutation of ^•O₂ ⁻ into O₂ and H₂O₂. Mitochondria are another source of ROS generation. Electrons are transferred along the ETC, finally inducing the reduction of O₂ to water at complex IV. VDACs control the release of ^•O₂ ⁻ from the mitochondria to the cytosol. Furthermore, H₂O₂ is also produced during protein folding in the ER. CoQ, coenzyme Q; Cyt C, cytochrome c; ER, endoplasmic reticulum; ERO1, endoplasmic reticulum oxidoreductin 1; ETC, electron transport chain; H₂O₂, hydrogen peroxide; NOX, nicotinamide adenine dinucleotide phosphate oxidase; ^•O₂ ⁻, superoxide anion; ROS, reactive oxygen species; SOD, superoxide dismutase; VDAC, voltage-dependent anion channel.

Seven NOX isoforms have been identified, namely, NOX1–5 and dual oxidase (DUOX) 1–2; these isoforms are differentially expressed in diverse cells and species, are located at distinct sites in the cell, and produce certain kinds of ROS. Specifically, NOX1–3 and NOX5 produce ^•O₂ ⁻, whereas NOX4 and DUOX1–2 produce H₂O₂ (Bedard and Krause, 2007). Specifically, NOX1–3 and NOX5 produce ^•O₂ ⁻, whereas NOX4 and DUOX1–2 produce H₂O₂ (Vermot et al., 2021).

In addition, mitochondria are another source of endogenous ROS. The primary site of mitochondrial ROS (mtROS) generation is the electron transport chain (ETC). The sites of ^•O₂ ⁻ formation are mainly Complex I (ubiquinone oxidoreductase) and Complex III (ubiquinol–cytochrome c oxidoreductase), which leak free electrons that drive monoelectronic O₂ reduction to ^•O₂ ⁻. Complex II (succinate dehydrogenase) also participates in ^•O₂ ⁻ formation. Subsequently, ^•O₂ ⁻ is rapidly converted into H₂O₂ by superoxide dismutases (SODs), and H₂O₂ is further decomposed by peroxiredoxins (Prxs) or glutathione peroxidases (GPXs) into H₂O (Islam et al., 2022) (Fig. 1).

ROS balance

At steady state, the level of ROS of any type is determined by the rate of ROS production relative to the rate of ROS scavenging. Excessive ROS formation may exacerbate nonspecific oxidative injury to a variety of cellular biomolecules, causing injuries such as gene mutations, DNA damage, biofilm lipid peroxidation, or protein denaturation (Wang et al., 2021b). As a result, sophisticated antioxidant mechanisms to eliminate excessive ROS formation have been developed (Wild, 2012), thereby lessening the negative effects of redox stress and preserving the equilibrium of ROS generation and clearance (Cadenas, 1997) (Fig. 2).

FIG. 2.

Key inducers and scavengers of ROS. The major inducers that promote ROS production are shown. To adapt to the hypoxic environment, cells produce a substantial amount of ROS. Some tumor-associated gene mutations positively correlate with the abundance of ROS. In addition, ER stress and exogenous factors stimulate redox reactions. Sophisticated antioxidant mechanisms to eliminate the negative effects of excess ROS have been developed. Antioxidant enzymes and the NRF2-KEAP1 systems could inhibit ROS generation. In addition, many tumor suppressor genes, such as FOXO, RB, P16, P21, BRCA1, and BRCA2, exhibit antioxidant activity. Orally delivered compounds and supplements such as NAC, vitamin C, and vitamin E have been proven to reduce oxidative stress. BRCA1/2, breast cancer susceptibility 1 and 2 genes; FOXOs, forkhead box proteins of the class O subgroup; GSH/GPX, glutathione/glutathione peroxidase; HIF-1, hypoxia-inducible factor-1; KEAP1, kelch-like ECH-associated protein 1; NAC, N-acetylcysteine; NCLX, mitochondrial Na⁺/Ca²⁺ exchanger; NRF2, nuclear factor erythroid 2-related factor 2; Prxs, peroxiredoxins; RB, retinoblastoma.

ROS inducers

Hypoxia

An important factor that promotes mtROS generation is hypoxia. Under acute hypoxic conditions, cells produce many ROS, which help them adapt to the hypoxic environment (Zhao et al., 2022). ROS production mainly originates from mitochondrial Complex I and Complex III in a hypoxic environment. Knocking down Complex I inhibited mitochondrial Na⁺/Ca²⁺ exchanger (NCLX) activity, and NCLX was required for hypoxia-induced increases in ROS levels. NCLX is critical for removing Ca²⁺ from mitochondria and for the influx of Na⁺ into mitochondria under hypoxic conditions, and inner mitochondrial membrane (IMM) mobility is affected by NCLX activity and mitochondrial Na⁺ concentrations (Hernansanz-Agustín et al., 2020). NCLX regulates ROS production by modulating the fluidity of the IMM.

Gene mutations

Gene mutations are associated with increasing intracellular ROS levels. Deactivation of tumor suppressor genes (Vurusaner et al., 2012; Weinberg et al., 2010; Zhu et al., 2014), activation of oncogenes (Ogrunc et al., 2014), and mutations in mitochondrial DNA (Dasgupta et al., 2012) are mechanisms that can lead to increased ROS production. For example, the tumor suppressors P53, P21, P16, forkhead box proteins of the class O subgroup (FOXOs), retinoblastoma (RB), and breast cancer susceptibility 1 and 2 genes (BRCA1/2) have been shown to regulate ROS levels (Vurusaner et al., 2012). The ectopic expression of Myc oncogenes, such as components of the mitogen-activated protein kinases (MAPK) and phosphoinositide 3-kinase (PI3K) signaling pathways, promotes oxidant production in the mitochondria and in other organelles (Maya-Mendoza et al., 2015; Murphy et al., 2008; Weinberg et al., 2010). The instability of mitochondrial DNA also contributes to increased ROS production (Woo et al., 2012).

Endoplasmic reticulum stress

The endoplasmic reticulum (ER) interacts with mitochondria, and stress-induced calcium release from the ER may stimulate mitochondrial bioenergetics leading to the overproduction of ROS (Bravo et al., 2012). ROS are also products of protein folding. The correct folding and posttranslational modification of nascent proteins in the ER depend on redox reactions. Endoplasmic reticulum oxidoreductin 1 (ERO1) catalyzes the reoxidation of protein disulfide isomerases, and each disulfide bond that forms during oxidative folding can produce a single ROS molecule (Shimizu and Hendershot, 2009). Misfolded nascent proteins can lead to ER stress and the unfolded protein response (UPR), which eventually result in the intracellular accumulation of ROS (Chen and Cubillos-Ruiz, 2021; Zhang et al., 2017).

Extracellular factors

Chronic exposure to numerous environmental toxins causes ROS levels to exceed physiological thresholds, which leads to mitochondrial damage, unstable energy metabolism, and an imbalanced redox state (Miller and Jones, 2014). Additional extrinsic factors have been shown to be exogenous sources of ROS, including air pollutants, cigarette smoke, ultraviolet radiation, γ-radiation, and several drugs (Nathan and Cunningham-Bussel, 2013).

ROS scavengers

Antioxidant enzyme system

SODs are the major antioxidant enzymes that catalyze the dismutation of ^•O₂ ⁻ to yield H₂O₂ (Miao and St Clair, 2009). In eukaryotes, three distinct SODs (SOD1, SOD2, and SOD3) are located primarily in the cytoplasm, mitochondrial matrix, and extracellular space, respectively (Hempel et al., 2011). The reduction of H₂O₂ into H₂O limits ^•O₂ ⁻ formation by the Fenton reaction; this reduction is regulated by both the catalase and peroxidase systems, such as the thioredoxin/thioredoxin reductase/peroxiredoxin (Trx/TrxR/Prx) and glutathione/glutathione peroxidase (GSH/GPX) systems (Weydert and Cullen, 2010). NADPH, which is essential for the activity of these antioxidant defense mechanisms, is an electron donor that facilitates reductase regeneration of GSH and Trx (Ju et al., 2020).

NRF2–KEAP1 system

The kelch-like ECH-associated protein 1 (KEAP1)-nuclear factor erythroid 2-related factor 2 (NRF2) system is a crucial participant in the defense against oxidative stress and the maintenance of redox homeostasis in eukaryotes. KEAP1 suppresses NRF2 activity under unstressed conditions, and NRF2 is liberated from KEAP1 upon exposure to oxidative stress (Yamamoto et al., 2018). KEAP1 is a cysteine thiol-rich major biosensor of electrophiles and ROS, whereas NRF2 is a key transcription factor involved in the expression of a number of antioxidant defense proteins, including the GSH biosynthesis genes glutamate-cysteine ligase modifier subunit (GCLM) and glutamate-cysteine ligase catalytic subunit (GCLC), the cystine transporter solute carrier family 7 member 11 (SLC7A11), and NADPH-generating enzymes such as malic enzyme 1 (ME1), isocitrate dehydrogenase 1 (IDH1), and glucose-6-phosphate dehydrogenase (G6PD) (Itoh et al., 1999; Mitsuishi et al., 2012; Moi et al., 1994; Thimmulappa et al., 2002; Wild et al., 1999).

Tumor suppressors

Tumor suppressor genes have been implicated in the regulation of diverse cellular activities, including activating the expression of certain antioxidant genes in response to oxidative stress. For instance, the activities of FOXOs and peroxisome proliferator activated receptor γ coactivator-1α (PGC-1α) contribute to the cellular redox status, which has been implicated in carcinogenesis (Brown and Webb, 2018; Guo et al., 2018). In addition, RB, P16, P21, BRCA1, and BRCA2 play key roles in regulating ROS levels (Vurusaner et al., 2012). Intriguingly, the tumor suppressor P53, which is activated by ROS and regulated via the redox status of cancer cells, may play both pro-oxidant and antioxidant roles (Maillet and Pervaiz, 2012).

P53 reduces ROS levels by increasing NADPH production by upregulating TP53-induced glycolysis and apoptosis regulator (TIGAR), which is a fructose-2,6-bisphosphatase that decreases the glycolysis rate and increases metabolic flux through the oxidative arm of the pentose phosphate pathway (PPP) (Cheung et al., 2013). In contrast, P53 exerts an effect similar to that of an ROS inducer by upregulating the expression of P53-inducible gene 3 (PIG3), which can lead to ROS accumulation by activating the NOX2 complex (Italiano et al., 2012).

Antioxidant compounds

In addition to the antioxidant enzyme system, orally ingested compounds and supplements have been reported to inhibit oxidative stress; these include N-acetylcysteine (NAC), vitamin C, vitamin E, β-carotene, and polyphenols (Bouayed and Bohn, 2010). Notably, NAC is a precursor of reduced GSH and an active scavenger of oxygen free radicals in vivo. Vitamin C is an enzyme cofactor and reductant that is concentrated in cells (Wilson, 2005). The vitamin E family includes four tocopherols and four tocotrienols, and their best-characterized function is as chain-breaking antioxidants that prevent the cyclic propagation of lipid peroxidation (Brigelius-Flohé and Traber, 1999).

Redox regulation in tumorigenesis

ROS may function to modulate tumorigenesis through diverse signaling pathways, and many of the aforementioned tumor-promoting events also promote ROS production. Because of their elevated ATP demands, tumor cells accumulate higher levels of ROS than their normal counterparts. As a result of ROS accumulation, DNA damage, genomic instability, and protein structure modification are essential for tumorigenesis (Fig. 3). Modulation of the epigenetic regulation of gene expression by modifying the activity of DNA methyltransferases (DNMTs) or histone deacetylases (HDACs) or hypomethylation due to oxidized DNA damage can induce tumor formation (O'Hagan et al., 2011).

FIG. 3.

Redox-dependent pathways that promote tumorigenesis. ROS can induce DNA damage, protein modification, and genomic instability, leading to the acquisition of potentially oncogenic mutations. ROS accumulation can induce tumor formation via the activation of oncogenic signaling pathways, the modulation of epigenetic regulation of gene expression by modifying the activity of DNMTs or HDACs, or by hypomethylation due to oxidized DNA damage. While ROS typically exhibit tumorigenesis-promoting activity, elevated levels of ROS may also prevent tumorigenesis by increasing oxidative damage and promoting ROS-dependent cell death. DNMTs, DNA methyltransferases; HDACs, histone deacetylases.

ROS may also promote tumorigenesis via the reversible oxidation of cysteine residues in thousands of proteins, a process that involves mediators in numerous signaling pathways associated with tumorigenesis (van der Reest et al., 2018). For example, RAC1, an ROS regulator with well-characterized oncogenic activity, is a small GTPase that drives malignant cell behavior, in part, by promoting NOX activation at the plasma membrane (Ellenbroek and Collard, 2007). RAC1 is activated after loss of the tumor suppressor adenomatous polyposis coli (APC) gene and facilitates colorectal cancer (CRC) initiation, which depends on the production of ROS mediated through NOX1 (Myant et al., 2013).

RAC1B, an alternatively spliced form of RAC1, drives malignant transformation of mammary epithelial cells through DNA damage and genomic instability caused by increasing mtROS levels (Radisky et al., 2005). RAC2 GTPase may also generate high levels of ROS by altering the mitochondrial membrane potential and electron flow through mitochondrial respiratory chain Complex III, thereby promoting the acquisition of oncogenic phenotypes in myeloid leukemia (Nieborowska-Skorska et al., 2012). In addition, mitochondrial oxidative stress drives malignant transformation, proliferation, and metastasis in a variety of tumors (Kuo et al., 2022). For example, mitochondrial oxidative stress induced by high expression of mitochondrial Lon promotes the epithelial–mesenchymal transition (EMT) through activation of ROS-dependent P38 and NF-κB signaling and the activation and polarization of M2 macrophages in the TME (Kuo et al., 2020).

Other studies have shown that increased levels of mtROS by Lon promote the malignant behavior of cancer cells through the MAPK and Ras-extracellular signal-regulated kinase (Ras-ERK) signaling pathways (Cheng et al., 2013; Weinberg et al., 2010). Moreover, mtROS promote the acquisition of a tumor-permissive phenotype and cause stress-induced genomic instability in breast cancer (BC) and other cancers due to sirtuin 3 (deacetylase in mitochondria) deficiency; this stress makes cells more susceptible to transformation (Kim et al., 2010). Another recent investigation showed that mitochondrial Complex I plays a key role in supporting the survival and proliferation of T cell acute lymphoblastic leukemia cells by generating mtROS (Kong et al., 2020). The loss of function that limits ROS production, such as deficiency in peroxiredoxin 1 (PRDX1), SOD1, or SOD2, can promote tumorigenesis by increasing the degree of oxidative damage to DNA (Busuttil et al., 2005; Rani et al., 2012; Van Remmen et al., 2003).

However, the mechanisms by which ROS promote tumorigenesis by damaging DNA and altering protein structures are not infinite. In contrast to the tumorigenesis-promoting activity, elevated levels of ROS may prevent tumorigenesis by increasing oxidative damage and enhancing ROS-dependent stress signaling. For instance, ferroptosis is a nonapoptotic form of cell death that is induced by iron-dependent lipid peroxidation resulting from ROS production, and ferroptosis leads to the elimination of tumor cells and thereby reduces malignancy development (Jiang et al., 2021b). The inhibition of ferroptosis by GPX4/GSH, which prevents the iron-mediated reactions of peroxides, may help maintain tumor cell survival and thereby establish a highly therapy-resistant high-mesenchymal cell state (Hangauer et al., 2017; Viswanathan et al., 2017).

Redox-Sensitive Transcription Factors in Cancer Metastasis

Extensive studies have suggested that a variety of redox-sensitive transcription factors, such as NRF2, hypoxia-inducible factor-1α (HIF-1α), and FOXOs, regulate redox signaling (Fig. 4). These factors are highly sensitive to ROS and play roles in maintaining redox homeostasis and regulating cancer metastasis. During cancer metastasis, malignant cells leverage these transcription factors to form complex defense systems that maintain the reductive environment needed to counteract stresses induced by rapid proliferation and a hostile microenvironment.

FIG. 4.

Redox-sensitive transcription factors that regulate cancer metastasis. ROS act on a plethora of redox-sensitive transcription factors, thereby eliciting diverse biological activities. These activities are highly pleiotropic and involve the antioxidant response, the hypoxic response, and stress adaptation. PHD, prolyl hydroxylase domain.

Nuclear factor erythroid 2-related factor 2

NRF2 is the master regulator of the cellular transcriptional program triggered by the antioxidant response; NRF2 helps maintain low basic protein levels during unstressed conditions but increases the expression of key genes under pathological conditions. Thus, NRF2 is involved mainly in promoting the production of a battery of endogenous antioxidant enzymes suitable for the reduction of ROS and the attenuation of oxidative stress. Mechanistically, ROS lead to conformational changes in KEAP1, an NRF2 inhibitor, thereby interfering with NRF2 ubiquitylation and resulting in NRF2 release from KEAP1. Subsequently, NRF2 accumulates in the cytosol and translocates into the nucleus, where it stimulates the constitutive activation and production of antioxidant enzymes (Baird et al., 2013).

Many studies have highlighted a key metastasis-promoting role for NRF2 in cancer cells. In a mouse model of lung adenocarcinoma driven by KEAP1 loss of function, NRF2 accumulation promoted metastasis by inhibiting the heme- and ubiquitin ligase Fbxo22-induced degradation of BACH1 (Lignitto et al., 2019). Interestingly, ROS-NRF2 activation also induced glycolysis, increased HK2 and GAPDH expression, and promoted lung cancer cell migration in a BACH1-dependent manner (Wiel et al., 2019). Furthermore, aberrant NRF2 activation by antioxidant antidiabetic agents through the ROS-NRF2-HO-1 axis contributed to an increase in metastasis-associated gene expression and accelerated metastasis in BC (Li et al., 2021c).

In gastric cancer, mitochondrial GRIM-19 deficiency also facilitates metastasis through the oncogenic ROS-NRF2-HO-1 axis (Wang et al., 2021a). In addition, NRF2 is modulated by certain redox-sensitive microRNAs. For example, miR-132 binds the 3′UTR (untranslated region) of NRF2, and downregulation of miR-132 led to increased bladder cancer cell migration by elevating the expression of NRF2 (Mao et al., 2022); a similar effect was recapitulated by miR-142-5p (Xiao et al., 2021). Collectively, these findings suggest a crucial role of NRF2 in promoting tumor metastasis, and NRF2 is therefore a promising target for preventing cancer metastasis.

Hypoxia-inducible factor-1α

The HIF family of transcription factors is a major oxygen sensor that mediates cellular adaptation to hypoxic microenvironments. Hypoxia is a hallmark of the TME and common in solid tumors. Solid tumors are exposed to a hypoxic microenvironment at both the initial and advanced stages of tumor progression. HIF-1α, a major oxygen sensor and oxygen homeostasis regulator, induces a series of signaling cascades to suppress oxidative phosphorylation and ROS production in mitochondria, thereby mediating cellular adaptation to hypoxic microenvironments and supporting tumor cell proliferation (Semenza, 2012). Under well-oxygenated conditions, HIF-1α is hydroxylated by prolyl hydroxylase domain (PHD) proteins and factor inhibiting HIF (FIH). PHDs induce the hydroxylation of HIF-1α, and hydroxylated HIF-1α is recognized by the von Hippel‒Lindau (VHL) protein, which recruits a ubiquitin ligase that targets HIF-1α for proteasomal degradation.

FIH inhibits the interaction of the HIF-1α subunit with the nuclear coactivators CBP/p300 and thus inhibits the transcription of HIF-dependent genes (Wang et al., 2022b). However, under hypoxic conditions, elevated generation of ROS contributes to HIF-1α stabilization and activation via various mechanisms. Specifically, ROS suppress the activity of PHDs and FIH in the cytoplasm and FIH is more sensitive to ROS than PHDs (Masson et al., 2012). In addition, ROS are also involved in activating NF-κB, an important transcription factor in the full activation of HIF-1α (Korbecki et al., 2021). Therefore, HIF-1α accumulates in the nucleus and forms a stable complex with HIF-1β and the p300 coactivator protein, which together activate the transcription of multiple hypoxia-responsive genes (Lee et al., 2020).

HIF-1α accumulation is common in cancer, indicating that hypoxic conditions may contribute to multiple stages of tumorigenesis. Increased HIF-1α protein levels have been associated with an increased risk of cancer mortality. HIF-1α regulates multiple pathways in cancer biology involving hypoxic processes, including EMT (Chen et al., 2021; Jan et al., 2019), glucose metabolism, stem cell maintenance, and cancer cell metastasis and invasion; thus, HIF-1α plays vital roles at certain stages of tumor progression. Several studies have demonstrated a direct role for HIF-1α activation in cancer cells, such as squamous, liver, lung, and CRC cells, leading to enhanced tumor metastasis (Fu et al., 2021; Hou et al., 2020; Hu et al., 2020; Song et al., 2019).

In line with these reports, mice in which the expression of HIF-1α was reduced via CRISPR/Cas9 technology had significantly inhibited pancreatic tumor growth and metastasis, which was accompanied by prolonged survival (Li et al., 2019a). C-X-C motif chemokine receptor 4 (CXCR4) is a downstream factor of HIF-1α and is also involved in promoting cell migration and invasion (Devignes et al., 2018; Ding et al., 2020; Wu et al., 2022). HIF-1α increases renal cell carcinoma (RCC) metastasis by forming a feed-forward loop with CXCR4 (Bao et al., 2019).

In addition, the roles of HIF-1α in the TME were also studied. For example, the increase in HIF-1α expression in cancer-associated fibroblasts (CAFs) stimulated CAF proliferation and migration by blunting the activity of miR-210, thereby suppressing the expression of vacuole membrane protein 1 (VMP1) (Yang et al., 2021). Another study showed that elevated tumor-associated macrophage (TAM)-associated HIF-1α expression was linked to a high tumor grade, increased metastasis, and poor overall survival in CRC patients (Cowman et al., 2020). Furthermore, an interferon-driven inflammatory TME promoted tumorigenesis and metastasis through upregulation of HIF-1α expression via JAK, RAS, PI3K/AKT/mTOR, and MAPK-P38 signaling cascades (Yeh et al., 2018).

Forkhead box proteins of the class O subgroup

The FOXO family is an important participant in the cellular stress response and promotes cellular antioxidant defense. The FOXO family includes the following four members: FOXO1, FOXO3, FOXO4, and FOXO6 (Benayoun et al., 2011). The transcription of FOXO genes is regulated by pathological stress stimuli that are frequently associated with ROS. ROS may modulate FOXO activity through posttranslational modifications, including phosphorylation, acetylation, and ubiquitination. These modifications may affect FOXO interactions with coregulators and alter their subcellular localization and stability, which are very complex and, in part, controversial (Klotz et al., 2015). Specifically, under elevated levels of ROS condition, several kinases are found to phosphorylate FOXO proteins and have been shown to stimulate or inhibit the transcriptional activity of FOXOs. For example, Akt, ERK, and p38 MAPK induce FOXO phosphorylation and usually result in FOXO inactivation and nuclear exclusion.

However, AMP-activated protein kinase (AMPK), c-Jun N-terminal kinase (JNK), and mammalian STE20-like kinase-1 (MST1) may phosphorylate and activate FOXO by stimulating its nuclear accumulation (Storz, 2011). In addition, ROS also induce the formation of heterodimers between p300/CBP acetylases and FOXO4 through intermolecular disulfide bridges linking redox-sensitive cysteine residues (Dansen et al., 2009). The acetylation state of FOXOs can either increase or inhibit their transcriptional activity depending on the FOXO isoforms and their transcriptional coactivators (Farhan et al., 2017). Moreover, the lysine residues in FOXO proteins can also become ubiquitinated under oxidative stress. A study showed that FOXO4 can be mono-ubiquitinated at Lys199 and Lys211, resulting in nuclear translocation and increased transcriptional activity in response to oxidative stress (van der Horst et al., 2006).

Recent studies have demonstrated important roles for FOXOs in maintaining redox homeostasis in tumors, and have shown that FOXOs suppress cell migration and metastasis. Other reports in the literature have suggested that FOXOs play an oncogenic role (Jiramongkol and Lam, 2020). The reason for this difference may be related to the ROS levels in these studies. The role of FOXO1 in suppressing cancer metastasis was supported by a study showing that CCL18 promoted the proliferation, migration, and invasion of intrahepatic cholangiocarcinoma (ICC) by inhibiting FOXO1, and the effect was reversed by the knockdown of a predominant receptor of CCL18 (Wang et al., 2022a). FOXO3a deficiency has been associated with poor clinical outcomes in melanoma (Yan et al., 2016), pancreatic cancer (Yan et al., 2017), and RCC (Ni et al., 2014) because its loss of function facilitates the proliferation and metastasis of cancer cells.

However, FOXO3a is not solely a tumor suppressor; elevated FOXO3a expression leads to increased lymph node metastasis and cancer cell migration in BC (Rehman et al., 2018).

Although overwhelming evidence has indicated that FOXO is a tumor suppressor, some literature reports have suggested that FOXO is a potential oncogenic protein. Elevated expression of FOXO6 in CRC and esophageal carcinoma has been correlated with poor prognosis and enhanced cancer metastasis (Li et al., 2021b; Li et al., 2019b). An oncogenic role of PAX3-FOXO1 in alveolar rhabdomyosarcoma tumorigenesis was also observed (Hu et al., 2021; Nguyen and Barr, 2018). In addition, the tumor-promoting abilities of FOXO1 have been discovered in the TME. FOXO1 was identified as an essential factor in mediating the migration of M2 macrophages via CCL20 secretion in esophageal squamous cell carcinoma (Wang et al., 2020). Thus, FOXOs can both suppress or support tumor growth and metastasis in a context-dependent manner.

Redox Regulation in the TME

Tumor cells grow in a complex microenvironment, where they construct a protective niche that favors tumor initiation, resistance, angiogenesis, and metastasis and ultimately leads to therapeutic failure. The TME is a multicellular system that is characteristic of all the noncancerous components in the tumor cell surroundings; these noncancer cells include endothelial cells, CAFs, TAMs, myeloid-derived suppressor cells (MDSCs), lymphocytes, the extracellular matrix, and other components. Although ROS in the TME are toxic to most cells, these cells also produce ROS to regulate a range of biological activities in cancer (Fig. 5). Therefore, a full understanding of the functional importance and molecular mechanisms involved in ROS regulation in the TME is a critical prerequisite for developing more effective targeted therapies against cancer.

FIG. 5.

Redox regulation in the TME. The production and regulation of ROS in TME-associated cancer and stromal cells contribute to the premetastatic niche and promote tumor metastasis. (A) Increased ROS production results in CAF activation. (B) Extracellular ROS production is necessary for TAM M2 polarization, and TAM-produced ROS promote cancer progression. (C) MDSCs suppress T cells and induce tumor immune escape through increased ROS production. (D) ROS signaling drives the metastasis of cancer cells through EMT. CAFs, cancer-associated fibroblasts; EMT, epithelial–mesenchymal transition; MDSCs, myeloid-derived suppressor cells; NFs, normal fibroblasts; TAMs, tumor-associated macrophages; TME, tumor microenvironment.

Redox regulation in CAFs

CAFs, constituting the largest subpopulation of stromal cells in the TME, exhibit enhanced tumor cell proliferative and migratory properties by secreting large quantities of growth factors, proinflammatory cytokines, and chemokines and producing multiple matrix proteins that remodel the extracellular matrix (Kalluri and Zeisberg, 2006). However, normal fibroblasts are initially quiescent and exhibit inhibitory effects on tumor cell proliferation and motility (Alkasalias et al., 2014). Therefore, normal fibroblasts undergo an education process that involves recruitment and reprogramming into protumorigenic CAFs by a broad range of cancer-derived factors, including transforming growth factor (TGF-β), platelet-derived growth factor (PDGF), IL-6, and specific TME stimuli, such as matrix stiffness, localized hypoxia, and oxidative stress (Chen and Song, 2019). Numerous studies have demonstrated that activated CAFs contribute to cancer metastasis (Kalluri and Zeisberg, 2006). Recently, attention has been directed to the roles of ROS in the CAF activation process, as ROS may ultimately promote cancer metastasis.

The critical role of ROS in promoting myofibroblast differentiation is widely recognized. In a chronic oxidative stress model, JunD^−/− -derived fibroblasts exhibited myofibroblastic features after HIF-1α accumulation and stimulated the CXCL12/CXCR4 signaling axis, which led to a high rate of mammary adenocarcinoma metastasis (Toullec et al., 2010). Tumor epithelial cell-produced H₂O₂ also participated in monocyte-to-myofibroblast transdifferentiation in a time- and concentration-dependent manner by activating the p38-MAPK pathway in pancreatic cancer (Huang et al., 2020). In addition, the participation of the oxidant agent H₂O₂, an inducer of the endothelial cell transition into myofibroblasts, contributed to the secretion of TGF-β1 and TGF-β2 and p38-MAPK phosphorylation, whereas blocking TGF-β reversed these outcomes (Montorfano et al., 2014). Increased NOX4-derived ROS production was involved in TGF-β1-stimulated kidney myofibroblast activation through the RhoA/ROCK pathway (Sampson et al., 2018). In contrast, NOX4 inhibitor supplementation attenuated TGFβ1-driven prostate fibroblast activation.

These results demonstrate that ROS are required for TGF-β signaling (Manickam et al., 2014). Furthermore, increased ROS generation in lung fibroblasts resulting from uncoupling protein-2 (UCP2) activation has been shown to promote myofibroblast differentiation and senescence by altering the cellular redox state (Rangarajan et al., 2022). Similarly, the effect of IκB kinase β loss of function in fibroblasts resulted in fibroblast–myofibroblast transformation and senescence by activating the stress-sensitive transcription factor AP-1/c-Jun and upregulating Tgfβ2, which ultimately accelerated cell migration and ROS accumulation (Chen et al., 2016).

Taken together, these data indicate that increased ROS production is a key event in myofibroblast differentiation in human tumors, which generates a protective niche to boost tumor development. Targeting the redox regulation of CAFs presents a promising approach to cancer therapy.

Redox regulation in TAMs

TAMs, the most abundant immune cells in the TME, are mainly classified as tumor-inhibiting M1 phenotype or tumor-promoting M2 phenotype macrophages (Pathria et al., 2019). The infiltration of M2 TAMs has been positively associated with malignant tumor progression and immune escape through the secretion of immunosuppressive cytokines, epidermal growth factor, and matrix metalloproteinases (MMPs); furthermore, M2 TAMs have been described as inhibitors of inflammatory responses, and promoters of tumorigenesis, angiogenesis, and metastasis in most types of human cancers (Chen et al., 2019b).

Numerous studies have highlighted extracellular ROS production in the TME, showing that it is necessary for recruiting and triggering the M2 polarization of macrophages and immune responses (Zhang et al., 2013). Oxidative stress-induced pancreatic cancer cell-derived release of exosomal KRAS^G12D protein was taken up by macrophages and caused the M2 polarization of macrophages and fatty acid oxidation via the signal transducer and activator of transcription 3 (STAT3) pathway, thus promoting tumor growth in vivo (Dai et al., 2020). In lung cancer, tumoral NOX4 stimulated macrophage migration and switching to a protumor M2-like phenotype by elevating JNK activity and releasing HB-EGF, which promoted the proliferation of cancer cells (Zhang et al., 2019). Another study showed that tumoral ROS induced M2 macrophage phenotype acquisition and enhanced the release of TNF-α through the MAPK/ERK pathway, thus facilitating malignant melanoma cell migration and invasion (Lin et al., 2013).

Moreover, suppression of ROS by antioxidants such as caffeic acid and MitoQ or inhibition of ER stress effectively blocked M2 TAM activity and partially induced their switch to the M1 phenotype, further boosting lymphocyte proliferation and inhibiting tumor cell proliferation and survival (Formentini et al., 2017; Jiang et al., 2021a; Orsolic et al., 2016). Hence, increased levels of exogenous ROS might present a mechanism by which tumors trigger the M2 polarization of macrophages.

In addition to being constantly confronted with extracellular ROS, the release of ROS is a major mechanism through which TAMs promote the acquisition of malignant phenotypes of tumor cells. Steady-state production of ROS by TAMs is upregulated in a variety of tumor models and in human cancer. In vitro coculture assays have shown that TAMs expressing NOXs enhanced the proliferation of CRC (Luput et al., 2017). The role of macrophage-produced ROS in the liver was further elucidated by macrophage-specific knockout NOX1 mice, which showed that NOX1 depletion attenuated tumor cell proliferation and slowed hepatocellular carcinoma (HCC) progression with decreased activation of the STAT3 and ERK signaling pathways (Liang et al., 2019).

In a peritoneal ovarian cancer metastasis mouse model, peritoneal residential macrophages that expressed high levels of T cell immunoglobulin and mucin domain-containing 4 (Tim-4) protein were characterized as Tim-4⁺ TAMs, and this subpopulation contributed to peritoneal ovarian cancer metastasis by producing higher levels of mitochondrion-related ROS than Tim-4⁻ TAMs (Xia et al., 2020). Nevertheless, the precise molecular mechanism by which TAM-produced ROS contribute to cancer progression appears to be highly context dependent and remains to be further elucidated.

Redox regulation in MDSCs

MDSCs, major immune-suppressing cells, induce antigen-specific CD8⁺ T cell tolerance, which leads to the escape of tumor cells from immune surveillance. The promotion of tumor cell growth and disease progression by MDSCs has recently been described in a variety of murine tumor models and in human cancers (Li et al., 2021a). The number of MDSCs typically increases in oxidative stress-prone environments under many pathologic conditions (Hegde et al., 2021); increased ROS production is a general characteristic of tumor MDSCs, contributing to their enhanced immunosuppressive properties and the maintenance of their undifferentiated state (Corzo et al., 2009; Ohl and Tenbrock, 2018). In liver cancer, MDSCs were significantly elevated and suppressed T cell proliferation through ROS production and arginase 1 (Arg-1) activation (Nan et al., 2018).

Similarly, MDSCs derived from patients with oral squamous cell carcinoma presented strong immune-suppressive effects via ROS production and the pSTAT3 pathway (Zhong et al., 2019). Both of these effects were reversed by ROS inhibitors. Moreover, tumor-induced MDSCs promoted the growth of CRC cells via cell-to-cell contact, which was abolished by specific nitric oxide (NO) and ROS inhibitors, and an expanded circulating MDSC population has been correlated with advanced TNM stages and lymph node metastases in CRC patients (OuYang et al., 2015).

NRF2 and HIF-1α are both involved in regulating the differentiation, survival, and immunosuppressive potency of MDSCs (Ohl and Tenbrock, 2018). NRF2 is critically involved in the MDSC response to extracellular ROS, as it promotes the production of H₂O₂ and helps maintain a steady-state level of circulating MDSCs in individuals (Beury et al., 2016). In addition, MDSC-secreted ROS enhanced circulating tumor cell (CTC) proliferation and metastasis via upregulation of Notch1 expression in tumor cells through the NRF2-ARE axis (Sprouse et al., 2019). Another study also observed that NRF2 activation increased the quantity and prolonged the survival of tumor-infiltrating MDSCs by balancing glycolysis and mitochondrial metabolism and reducing oxidative stress (Ohl et al., 2018). Notably, HIF-1α alters the fate and function of MDSCs in the hypoxic TME.

Studies with HIF-1α-deficient mice revealed an important role for HIF-1α in enhancing MDSC-mediated T cell suppression by increasing arginase activity and NO production and promoting MDSC differentiation into immunosuppressive TAMs (Corzo et al., 2010). In summary, these findings suggest that ROS play important roles in regulating MDSC-mediated immune suppression, which contributes to tumor progression through complex mechanisms.

In summary, ROS act as crucial signaling molecules not only in cancer cells but also in most stromal cells, such as CAFs, TAMs and MDSCs. As described in this section, the major effects of ROS in the TME are the conversion of fibroblasts into myofibroblasts, the polarization of TAMs into the M2 phenotype, and the enhancement of MDSC-mediated T cell suppression, all of which shape the TME. ROS production by different members of stromal cells can exhibit both protumor and antitumor behavior, which depends mainly on their level, location, and regulation. The complex picture emerges in which ROS stimulate tumor–stromal interactions, thus forming a positive feedback loop and promoting cancer metastasis. However, it must be acknowledged that high heterogeneity within the TME remains a key obstacle in fully understanding the role of ROS in cancer treatment.

Many studies rely heavily on different members of stromal cells in the TME, which are often difficult to compare with each other; therefore, important steps forward in future research are needed.

Redox Regulation During Tumor Metastasis

Metastasis is the leading cause of death in patients with various types of tumors (Lambert et al., 2017). As a tumor progresses, tumor cells may delaminate from their original tumor, invade nearby tissue, and migrate to new sites via the blood and/or lymph circulatory system. In contrast to cell proliferation and survival at a primary site, metastasis in a diverse and changing environment requires migratory, invasive, and circulatory survival mechanisms and the capacity for regrowth in the environment of a distant organ (Vanharanta and Massague, 2013). Therefore, different redox reactions may be initiated during different processes of tumor metastasis (Fig. 6). ROS signaling drives and promotes metastatic responses, including EMT and cancer cell migration, invasion, and adhesion to endothelial cells. However, excessive ROS accumulation due to oncogene activation, matrix shedding, or high ROS levels in the circulatory system can also lead to tumor cell death.

FIG. 6.

Redox regulation during tumor metastasis. Redox reactions regulate different processes of tumor metastasis. ROS act as signaling sensors that drive the formation of key structures such as invadopodia or pseudopodia and facilitate the migration and invasion of tumor cells from the primary site to the surrounding stroma. Then ROS entail the escape of tumor cells from the primary site into the circulation and lymphatic system. Few CTCs can survive the highest levels of oxidative stress in blood vessels, and they show a strong antioxidant capacity to resist tumor cell killing by ROS. In the lymphatics and lymph nodes, lower levels of ROS, fewer free iron ions, and higher levels of GSH and monounsaturated fatty acid oleic acid could inhibit tumor cell death. To achieve the final step to reach distant organs, cancer cells need to adapt to unfamiliar environments, including different types and/or levels of ROS and nutrients, and create permissive niches to proliferate at secondary sites. In general, ROS either promote or inhibit cancer metastasis depending on a different complex context. CTCs, circulating tumor cells.

In general, ROS can both promote and inhibit tumor metastasis, with the outcome determined, in part, by several factors; the key point is now identification of specific regulatory mechanisms and interactions.

Redox regulation in primary sites

The first step in tumor metastasis is the migration and invasion of cells from the primary tumor site to the surrounding stroma, in which actin cytoskeleton reorganization is continuously coordinated (Fares et al., 2020). Key structures such as invadopodia and pseudopodia are formed by this cytoskeletal rearrangement, and their formation depends on ROS signaling (Diaz et al., 2009; Gianni et al., 2010; Gianni et al., 2009). High ROS levels have been detected during the detachment of primary tumor cells, and ROS are critical for activating different types of MMPs, which degrade the extracellular matrix and thus aid primary tumor cell invasion into the surrounding tissue (Mori et al., 2019; Nelson and Melendez, 2004; Shinohara et al., 2010).

However, tumor cell detachment from the extracellular matrix during cell invasion results in changes in signaling pathway activation and metabolism, which increases oxidative stress (Hawk and Schafer, 2018; Jiang et al., 2016; Schafer et al., 2009). These increases in ROS levels may result from reduced ox-PPP flux and G6PD activation in cells, which enables anchorage-independent growth (Schafer et al., 2009; Zhang et al., 2021).

Another mechanism that allows anchorage-independent growth of detached tumor cells involves the modification of glutamine metabolism, which increases reductive carboxylation and supports mitochondrial NADPH production mediated by isocitrate dehydrogenase 2 (IDH2) (Bueno et al., 2019; Jiang et al., 2016). Whereas the functions of ROS that promote EMT and cancer cell invasion have been reported in several studies (Cho et al., 2014; Hudson et al., 2014; Kesanakurti et al., 2017), some recent studies have reported that inhibiting ROS production may support tumor cell dissemination. Two studies simultaneously revealed that the stabilization of BACH1 promoted glycolysis-dependent lung cancer metastasis and that the heme- and Fbxo22-mediated degradation of BACH1 occurred after NRF2 activation was inhibited (Lignitto et al., 2019; Wiel et al., 2019).

As a result, the ROS levels in the tumor cells in situ were dynamically regulated, showing a tendency for increased ROS levels in cells leaving the original tissue and reduced ROS levels in cells invading the surrounding tissue.

Redox regulation in circulation and the lymphatic system

The second step in metastasis is the escape of tumor cells from the primary site and their entry into the blood circulatory and lymphatic systems. CTCs show the highest levels of oxidative stress during metastasis because blood is an oxidizing environment (Tasdogan et al., 2020). Few CTCs survive this high oxidative stress, and they show high antioxidant capacity to resist killing by ROS through the upregulation of antioxidant enzymes and the reprogramming of cellular metabolism (Chen et al., 2019a; Tasdogan et al., 2020).

Ferroptosis marked by lipid oxidation is the key form of CTC death under oxidative stress conditions in the blood, and polyunsaturated fatty acids (PUFAs) in membrane phospholipids are oxidized by redox-active iron during ferroptosis (Dixon and Stockwell, 2019). Melanoma CTCs alter lipid oxidation by increasing the transcription of the iron carrier transferrin, which reduces the intracellular iron pool, ROS levels, and lipid peroxidation rate. Melanoma CTCs also increase the incorporation of monounsaturated fatty acids (MUFAs) into membrane lipids, which displaces PUFAs (Hong et al., 2021; Ubellacker et al., 2020). In melanoma, CTCs show higher ROS levels than primary tumor cells, but clinical trials have shown that supplementation with antioxidants in fact increases cancer risk.

A study with mice also showed that antioxidants accelerated CTC metastasis (Le Gal et al., 2015). Further research indicated that monocarboxylate transporter 1 (MCT1) was the crucial transporter with elevated expression in melanoma CTCs in response to increased ROS levels. MCT1 promoted the metastasis of melanoma cells by promoting circulating lactate uptake, and inhibition of MCT1 suppressed ox-PPP and NADPH activity and ROS levels (Reinert et al., 2006; Tasdogan et al., 2020). CTCs from various kinds of tumors have also been shown to overexpress β-globin (HBB), which enhances the CTC antioxidant response and mediates a cytoprotective effect during blood-borne metastasis. Downregulation of HBB expression in CTCs increased the apoptosis rate following increased oxidative stress and reduced lung metastasis, but this downregulation had minimal impact on the progression of the primary tumor (Zheng et al., 2017).

Another mechanism that promotes the metastatic spread of tumor cells is that both CTC clusters and CTC-neutrophil clusters contribute to the survivability of tumor cells via the reduction of CTC exposure to oxygen and mtROS production (Aceto et al., 2014; Szczerba et al., 2019).

In an alternative route of cancer cell dissemination, the lymphatic system, including lymph nodes, promotes the metastasis of tumor cells by expressing vascular endothelial growth factor (VEGF) and various chemokines (Das et al., 2013; Ma et al., 2018). Tumor cells in the lymph system survive more easily and form a higher number of metastases than tumor cells in the blood because the lymph system carries lower levels of ROS and free iron and higher levels of GSH and the MUFA oleic acid. As a result, tumor cells in the lymph system present with a different metabolic status compared with tumor cells in the blood.

For example, melanoma tumor cells in the lymph system were less sensitive to ferroptosis than tumor cells in the blood, and elevated oleic acid levels in lymph enhanced the metastatic potential of tumor cells by protecting them from ferroptosis induced by lipid oxidation in an ACSL3-dependent manner (Ubellacker et al., 2020). Moreover, the ability to oxidize fatty acids increases the likelihood that tumor cells can survive in the lymph system (Lee et al., 2019). Fatty acid transporters, including CD36, tend to be highly expressed in tumor cells in the lymph system, which enhances the metastasis of oral carcinoma, ovarian carcinoma. and BC by promoting palmitic acid uptake (Pascual et al., 2017).

Redox regulation in distant sites

The final step in metastasis involves tumor cells reaching distant organs, where they need to adapt to an unfamiliar environment; this adaptation includes exposure to different types and/or levels of ROS and nutrients and thus the creation of permissive niches to proliferate at secondary sites. Nascent metastases undergo oxidative stress, including elevated ROS levels and low GSH-to-oxidized GSH and NADPH-to-NADP⁺ ratios, although the extent of this oxidative stress varies between metastatic sites (Basnet et al., 2019; Piskounova et al., 2015). Successfully metastasized tumor cells seem to not fully reactivate the anabolic pathways required for tumor proliferation due to oxidative stress. Tumor cells may shut down some of the anabolic pathways in response to oxidative stress during metastasis. Adaptation to metabolic stress is critical for metastatic growth. AMPK activation, for example, could inhibit tumor cell death by redox regulation during metabolic stress.

AMPK activation maintains NADPH levels by inhibiting NADPH consumption in fatty acid synthesis and increasing NADPH production in fatty acid oxidation (Jeon et al., 2012). However, once the diameter of a metastatic tumor has reached a suitable size, the cancer cells may become sufficiently adapted to attenuate the oxidative stress state, thereby enabling extensive activation of anabolic pathways.

In summary, throughout metastasis, tumor cells undergo complex environmental changes, including changes in ROS levels caused by dysregulation of genes in tumor cells, as well as changes from environmental factors. ROS may play facilitating or inhibiting roles in the process of tumor metastasis. To fully understand the complex mechanisms of tumor cell metastasis, we need to decipher the multiple factors that control ROS production and the consequences of both tumor cells and stromal components. This may be the key issue in further studies that must be addressed to achieve targeted therapy for cancer by modulating ROS.

Therapeutic Strategy to Target ROS in Cancer Metastasis

It has been mainly accepted that ROS at moderately elevated levels are important signaling molecules in multistage carcinogenesis that activate prosurvival signaling pathways (Cheung and Vousden, 2022). Undoubtedly, both low and high levels of ROS can be deleterious to tumor metastasis and progression since they are unable to perform beneficial biological activities by activating prosurvival signaling pathways at low levels and cannot be regulated by the antioxidative system at high levels, which could result in genotoxicity and proapoptosis. Anticancer chemotherapeutic drugs have been shown to boost ROS generation and mitochondrial dysfunction, resulting in oxidative stress-induced cell death (Niu et al., 2021). Besides, a large population-based prospective study provided evidence that the supplementation of antioxidant selenium has an obvious preventive effect on pancreatic cancer (Han et al., 2013).

However, inhibition of ROS may also exert a harmful effect, and promote tumor progression and metastasis. Long-term supplementation with the antioxidants NAC and vitamin E could stimulate the metastasis of KRAS-driven lung cancer (Wiel et al., 2019). The emergence of seemingly contradictory studies in treatment is due to the complexities of ROS responses.

Previous studies have indicated that ROS play significant roles in the metastasis process involving a complex crosstalk between cancer cells and other stromal cells in the TME (Sorolla et al., 2021). Therefore, a large amount of medicines have been developed to inhibit cancer metastasis by interfering with ROS-mediated metastasis pathways in tumor or critical stormal cells in the TME. Drugs that modify ROS levels in cancer cells through diverse mechanisms could significantly suppress metastasis, but no straightforward association has been identified between the inhibition or elevation of ROS and an antimetastatic effect (Liao et al., 2019). Fangchinoline could effectively inhibit metastasis by suppressing the ROS-related AKT-mTOR signaling pathway through deregulation of cytosolic ROS production (Chen et al., 2022). Elemene nanoemulsions could inhibit BC metastasis by decreasing the ROS-mediated activation and stabilization of HIF-1α, which reduces angiogenesis (Han et al., 2021).

In contrast, high levels of intracellular ROS induced by certain agents, including decylubiquinone and ginsenoside-Rh1, have also been observed to exhibit potent anticancer effects on tumor metastasis by regulating mtROS-mediated angiogenesis and metastatic factors (Cao et al., 2020; Jin et al., 2021).

In addition, the involvement of ROS as the key instigating agent for the immunosuppressive effect of tumor-associated stromal cells in the TME is attracting attention (Liao et al., 2019). Targeting intracellular ROS in CAFs by curcumin and AC1MMYR2, a small-molecule inhibitor of miR-21, has been shown to inhibit CAF-induced EMT in prostate and BC (Du et al., 2015; Ren et al., 2016). Moreover, regarding the regulation of ROS derived from immune cells, simvastatin and fluoxetine produced an antitumor effect by reducing oxidative stress, and partially reversing the ROS-induced polarization of TAMs in BC (Alupei et al., 2015; Ghosh et al., 2015). Hence, targeting TAM-mediated oxidative stress and reprogramming the transition of TAMs into the antitumor M1 phenotype may be prospective strategies.

On the contrary, decreasing the number of MDSCs or accelerating the maturation of MDSCs by limiting ROS generation is also an ideal theoretical strategy (De Cicco et al., 2020). For instance, a nanodrug composed of the photosensitizer chlorin e6 (Ce6) and the PI3K inhibitor IPI-549 could inhibit CRC metastasis by stimulating the apoptosis of MDSCs and the maturation of dendritic cells via the downregulation of Arg-1 and ROS production in MDSCs (Ding et al., 2021). In addition, curcumin may also inhibit MDSC proliferation and function by suppressing IL-6 and immune-suppressive mediators such Arg-1 and ROS (Liu et al., 2016). Overall, these discoveries highlight the critical function of ROS in promoting metastatic reprogramming and reveal a potential therapeutic avenue to target ROS at the nexus of the TME and metastasis.

Notably, to tackle the low response rates of immunotherapy, the joint application of immune checkpoint blockade and targeted therapies has shown synergistic therapeutic efficacy (Yi et al., 2022). Combining ROS-modulating agents with immune checkpoint blockade may yield efficient suppression of tumor metastasis and prolong patient survival. For instance, miR-21-3p-loaded gold nanoparticles improved the IFN-γ-mediated ferroptosis sensitivity of cancer cells by enhancing lipid ROS generation and displayed a synergistic effect with an anti-PD-1 antibody (Guo et al., 2022). In addition, the combination of anti-PD-L1 immune checkpoint inhibitors (ICIs) and sonodynamic treatment, which is based on ultrasound activation of a sensitizer that promotes ROS generation, significantly inhibited tumor progression in a mouse model of bilateral pancreatic cancer (Nesbitt et al., 2021).

Accordingly, a novel cancer therapeutic strategy may be forthcoming based on the administration of ROS-mediated drugs in conjunction with immunotherapy. More preclinical experiments and clinical trials are needed to assess the effect of ROS-targeting therapy in conjunction with anti-PD-1/L1 ICIs in the future.

In summary, it is not advisable to develop universal therapeutics using a simplistic approach given the complexity and variability of ROS regulation. Continued efforts to understand ROS production and regulation in cancer and TME stromal cells, as well as how they interact, will help refine antitumor strategies. Furthermore, the combination of emerging ROS-regulating strategies and cancer immunotherapy will greatly enhance therapeutic efficacy.

Conclusion

ROS exhibit profoundly different properties, mechanisms, and roles in different cells, not only in cancer cells but also in stromal cells in the TME during cancer metastasis; thus, ROS present not only great challenges but also opportunities for novel approaches to clinical treatment. Overall, an important step forward involves targeting the intricate nature of ROS regulation in cancer metastasis, which will facilitate the development of novel therapeutics designed to not only prevent tumor initiation but also halt cancer progression and ultimately improve patient well-being and survival.

Footnotes

Acknowledgment

We thank Sagene eBioart for the help with pattern diagram making.

Authors' Contributions

Y.L. and X.Z., as corresponding authors, planned, conceptualized, and critically revised the article. W.L., B.W, and M.Z. wrote and edited the original article. D.L. and F.C. created the figures and revised the article. All authors contributed to the article and approved the submitted version.

Author Disclosure Statement

The authors declare no competing financial interests.

Funding Information

This work was supported by grants from the National Natural Science Foundation of China (Nos. 82273142 and 82073197).

Abbreviations Used

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