Microsomal glutathione transferase 1 in cancer and the regulation of ferroptosis

Abstract

Microsomal glutathione transferase 1 (MGST1) is a member of the MAPEG family (membrane associated proteins in eicosanoid and glutathione metabolism), defined according to enzymatic activities, sequence motifs, and structural properties. MGST1 is a homotrimer which can bind three molecules of glutathione (GSH), with one modified to a thiolate anion displaying one-third-of-sites-reactivity. MGST1 has both glutathione transferase and peroxidase activities. Each is based on stabilizing the GSH thiolate in the same active site. MGST1 is abundant in the liver and displays a broad subcellular distribution with high levels in endoplasmic reticulum and mitochondrial membranes, consistent with a physiological role in protection from reactive electrophilic intermediates and oxidative stress. In this review paper, we particularly focus on recent advances made in understanding MGST1 activation, induction, broad subcellular distribution, and the role of MGST1 in apoptosis, ferroptosis, cancer progression, and therapeutic responses.

1. Introduction

Glutathione transferases (GSTs), originally discovered as detoxification enzymes (Booth, Boyland, & Sims, 1961; Combes & Stakelum, 1961), are one of the key enzymes involved in the metabolism of endogenous and exogenous electrophilic molecules with hydrophobic character. The fundamental basis for all GST catalysis is the capacity to lower the pKa of the reduced glutathione (GSH) from 9.0 in aqueous solution to about 6.5 to promote thiolate (GS⁻) anion formation when GSH is bound in the G-site, and to bind hydrophobic electrophilic substrate in H-site, catalyzing the conjugation of GSH to the substrate (Armstrong, 1994; Hayes & Pulford, 1995; Nebert & Vasiliou, 2004). This function is shared by two entirely distinct superfamilies of enzymes, one microsomal and the other cytosolic. These two superfamilies are not evolutionarily related and are found both in mammals and insects. Cytosolic GST gene family comprises 16 genes in six subfamilies—alpha (GSTA), mu (GSTM), omega (GSTO), pi (GSTP), theta (GSTT) and zeta (GSTZ). The active cytosolic enzyme exists as a dimer of the two subunits. Mitochondrial Kappa (GSTK) can be considered as a third type of GST (Bresell et al., 2005; Nebert & Vasiliou, 2004).

Microsomal glutathione transferase 1 (MGST1) is a member of the MAPEG family (membrane associated proteins in eicosanoid and glutathione metabolism), defined according to enzymatic activities, sequence motifs, and structural properties (Jakobsson, Morgenstern, Mancini, Ford-Hutchinson, & Persson, 1999). MAPEG family consists of six human proteins including MGST1, MGST2, MGST3, 5-lipoxygenase-activating protein (FLAP), leukotriene C4 (LTC4) synthase and microsomal prostaglandin E₂ synthase 1 (MPGES1, earlier referred to as MGST1-L1). Human MGST1 is most closely related to MPGES1 and less similar to the other four human MAPEG proteins. MAPEG family use activated GSH in catalysis. Glutathione transferase activity can be regarded as a common denominator for a majority of MAPEG, including MGST1, MGST2, LTC4 synthase and MPGES1 subfamilies, whereas glutathione peroxidase (GPX) activity occurs in representatives from the MGST1, 2 and 3 and MPGES1 subfamilies. FLAP, LTC4 synthase and MPGES1 have specific roles in the production of LTC4 and prostaglandin E₂. MGST2 (Bresell, et al., 2005; Jakobsson et al., 2000) and MGST3 (Steinmetz-Spah et al., 2022) are involved in LTC4 production or prostaglandin as well. The structures of MGST1 (Holm et al., 2006; Kuang et al., 2017), MGST2 (Thulasingam et al., 2021), LTC4 synthase (Ago et al., 2007; Martinez Molina et al., 2007), FLAP (Ferguson et al., 2007) and MPGES1 (Jegerschold et al., 2008) were resolved, and the results confirmed that the MAPEG proteins exist as homotrimers, with the active site residing at subunit interfaces; a conserved arginine plays a crucial role in stabilizing the GS⁻ anion; and conjugation of GSH to substrate proceed in the hydrophobic binding pocket (Morgenstern, Zhang, & Johansson, 2011).

MGST1 is an abundant membrane-bound enzyme displaying both GST and GPX activities toward multiple substrates ranging from products of lipid peroxidation, halogenated hydrocarbons and carcinogenic drugs (Morgenstern et al., 2011). Arginine 130 plays an essential role in stabilizing the GS⁻ anion form of GSH for catalysis. Mutation of arginine 130 to alanine resulted in complete loss of activity (Kuang et al., 2017). MGST1 contains three active sites with different affinities for GSH and that only the high affinity site is catalytically competent. GSH initially binds to each of the three low affinity sites in MGST1 (Kd = 50 mM). Once one molecule of bound GSH is deprotonated (Kd = 20 μM), the remaining active sites bind their GSH more strongly (Kd = 2.5 mM for the third GSH), but cannot promote deprotonation. This results in one-third-of-sites-reactivity (Alander et al., 2009).

The expression of Mgst1 mRNA was found to be most abundant in the livers for both genders of the rat, mouse and canine. In addition, testis, lung, kidney and heart have relative high expression (Mattes, Daniels, Summan, Xu, & Mendrick, 2006). MGST1 was expressed at much higher levels in adult livers than in perinatal livers in mice (Lu et al., 2013). A marked increase with age was noted in human hematopoetic stem and progenitor cells (Prall et al., 2007). However, MGST1 was observed to decrease in middle- and old-age rat liver compared to young rats, consistent with the upregulation of miR-34a and miR-93 during aging and downregulation of their targets SP1 and Nrf2 which are the transcription factors for MGST1 (Li, Muthusamy, Liang, Sarojini, & Wang, 2011).

MGST1 has been reviewed previously (Morgenstern, 2005; Morgenstern et al., 2011). Here we will focus on recent advances made in understanding MGST1 activation, induction, broad subcellular distribution, and the role of MGST1 in apoptosis, ferroptosis, cancer progression, and therapeutic responses.

2. Subcellular distribution of MGST1

MGST1 displays a broad subcellular distribution with especially high levels in endoplasmic reticulum (ER) and outer mitochondrial membranes (OMM), constituting 3% and 5% of the membrane protein content in the respective compartments of rat liver (Morgenstern, 2005; Morgenstern, Lundqvist, Andersson, Balk, & DePierre, 1984). MGST1 contains four transmembrane segments (TMSs) per subunit, and the amphipathic character of the first TMS constitutes a molecular determinant for the dual targeting of MGST1 to both ER and mitochondria. The importance of the N-terminal hydrophobic sequence for ER targeting (Kunze & Berger, 2015; Meacock, Greenfield, & High, 2000), and the amphipathic sequences for mitochondrial localization (von Heijne, 1986) are well established. MGST1 contains an N-terminal hydrophobic sequence, forms an alpha-helix in the first TMS (Holm, Morgenstern, & Hebert, 2002) that contains a well-conserved charged residue, lysine-25, which is internally charge compensated by aspartic acid-78 in the second TMS. Removing the positive charge of lysine-25 promotes ER incorporation, but counteracts mitochondrial insertion. In contrast, introducing an extra lysine in the first TMS of MGST1 has the opposite effect (Shimoji et al., 2017). The enzyme has been detected in other organelle membranes including peroxisomes (Islinger, Luers, Zischka, Ueffing, & Volkl, 2006; Nishino & Ito, 1990) and the plasma membrane (Horbach, Sies, & Akerboom, 1993; Horbach, Sies, & Akerboom, 1994). The localization in different membranes might be of particular significance, as the reactive lipophilic substrates, which concentrate in the membranes, cannot redistribute freely. Broad MGST1 subcellular distribution is thus consistent with a physiological role in protection from reactive electrophilic intermediates and oxidative stress.

3. MGST1 activation

One of the quickest and most efficient ways to enhance enzyme activity is to enact modification of enzyme. MGST1, which contains one cysteine residue (Cys-49) per subunit, has low enzyme activity in its native form, but can be subsequently activated by exposure to sulfhydryl-reacting agents (Morgenstern, DePierre, & Ernster, 1979), thiol-disulfide exchange (Aniya & Anders, 1989), S-glutathionylation (Dafre, Sies, & Akerboom, 1996), S-nitrosylation (Imaizumi, Miyagi, & Aniya, 2006), sulfhydryl oxidation to sulfenic acid (Shinno et al., 2005) or protein dimerization (Aniya & Anders, 1992). The reactivity (stability) of the thiol in MGST1 is impacted by surrounding phospholipids including cardiolipin, a mitochondrial specific lipid that prevents MGST1 activation by thiol modification. MGST1 can also be activated by tyrosine nitration (Ji & Bennett, 2003) or proteolysis (Morgenstern et al., 1989). While specific information on mechanisms by which MGST1 activation proceeds are presently undefined, these are known to involve a higher rates of formation of the GSH thiolate anion within the activated enzyme (Andersson et al., 1994; Svensson et al., 2004).

Addition of acrolein (α, β-unsaturated aldehyde) to isolated rat liver microsomes can increase MGST1 activity 2–3-fold. At a relatively high exposure level, acrolein appeared to inhibit MGST1. Activation is due to adduct formation with Cys 49, and inactivation may involve adduct formation to other nucleophilic sites in amino acids within the enzyme. In MGST1 histidine, arginine and lysine residues are present in close proximity to the active site. The amino groups in arginine and lysine bind the carboxyl group of GSH. Adduction to amino groups could explain the inhibitory effects of acrolein. Preferential reactivity of acrolein with thiol over amino groups could explain why the enzyme is activated at a low acrolein levels and inhibited at high. These apparently opposing forms of direct adaptation on the level of enzyme activity further narrow the thin line between survival and promotion of cell death, governed in this case by the acrolein exposure rates (Sthijns et al., 2017).

4. MGST1 induction

The underlying need for MGST1 induction is thought to be an adaptive response to chemical or oxidative stress within the cell. Thus, understanding what controls the induction of MGST1 could be useful from a therapeutic standpoint and also as a biomarker. Modulation of MGST1 expression could be used to modulate the effectiveness of chemotherapeutic drugs. Therefore, knowledge gained from studies on the mechanisms of induction of this enzyme can be utilized in drug development. Upregulation after different treatments have been noted (see some of the examples below).

Under acute hypobaric hypoxia, a 6-fold induction in the transcript levels of Mgst1 was observed in murine heart, likely providing a protective role for intracellular membranes against oxidative stress (Karar et al., 2007).

In zebrafish and the Japanese rice fish (medaka), at least four genes, frh3 (ferrintin H3), mgst1 (microsomal glutathione S-transferase 1), cmbl (carboxymethylenebutenolidase homolog) and slc40a1 (solute carrier family 40 [iron-regulated transporter], member 1) were significantly upregulated after treatment of arsenic; thus, these four genes might be robust biomarkers for identification of arsenic toxicity across species (Xu, Lam, Shen, & Gong, 2013).

Besides CYP1A1 and NQO1, MGST1 is one of the xenobiotic metabolism genes that was up-regulated in both MCF-7 and HepG2 cells in response to chemical carcinogens benzo(a)pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin, likely to play roles in the cellular response, and represent biomarkers of exposure for these compounds (Hockley et al., 2007; Hockley, Arlt, Brewer, Giddings, & Phillips, 2006).

MGST1 was the top upregulated protein (6.8–7.4 fold) in Chlamydia trachomatis infected Hela-229 human cervical carcinoma epithelial cells. These cells may initiate an inflammatory response by producing MGST1 to protect from oxidative stress caused by the C. trachomatis infection (Tan et al., 2016).

In a long-term (15 weeks) high-fat and high-sucrose diet (HFHSD)-induced obesity and diabetes mouse model, multiple proteins involved in glucose catabolism and lipogenesis were downregulated, whereas proteins involved in oxidative phosphorylation and lipid peroxidation (including MGST1) were upregulated, further evidence of a response to oxidative stress. This response is, once again, a cellular response to neutralize ROS generated by HFHSD at week 15 and protects the liver tissue from damage and results in disease stabilization (Midha et al., 2012).

Hydroxyurea directly scavenges free radicals and induces the expression of antioxidant genes in human umbilical vein endothelial cells, such as SOD2, GSR, MGST1 (log fold change 1.733), GSTM2, CBR1 (carbonyl reductase 1) and KLB (klotho B) (Santana et al., 2020). Reducing the hemoglobin S autoxidation maybe one of the mechanisms for the effect of hydroxyurea on sickle cell anemia.

In elemental mercury (Hg)-exposed rat lung, the expression of genes encoding GSTP, MGST1, glutathione reductase, and thioredoxin peroxidase was enhanced, indicating the activation of GSH redox system as an adaptive response to overcome Hg-induced oxidative stress and detoxify Hg metabolites (Liu, Lei, Waalkes, Beliles, & Morgan, 2003).

The upregulation of liver genes for heme degradation (Hmox1 and Prdx1), iron cellular transport (Slc40a1), and GSH synthesis and utilization (Gclc and Mgst1 ~ 2-fold) were early markers of the methimazole metabolism induced oxidative stress response within the blood cells (Vickers, Sinclair, Fisher, Morris, & Way, 2010).

Short-term anesthesia with avertin or isoflurane led to the upregulation of Mgst1, Thbs4 (thrombospondin 4), and Syp (synaptophysin) that persisted for at least 4 days in mouse hippocampus. Prolonged changes of these genes may help to protect the brain cells against neurotoxic substances generated after anaesthesia, and thus counter some of the toxic effects. This finding challenges the opinion that the effects of general anaesthesia, even for short periods, disappear from the brain within hours, and indicates that general anaesthesia should be used with caution and evaluated further to understand both short- and long-term risks associated with its use (Pekny, Andersson, Wilhelmsson, Pekna, & Pekny, 2014).

Negative results in paraquat-treated keratinocytes and downregulation in parathion/estrogen-treated breast cells were also obtained. The herbicide paraquat undergoes redox cycling generating reactive oxygen intermediates in both undifferentiated and differentiated mouse keratinocytes. mRNA expression of GSTAs was markedly increased in both cells in response to paraquat treatment. GSTP1 levels increased 3–4-fold following paraquat treatment. However, no alterations in MGST1 expression were evident under paraquat (Black et al., 2008b) or UVB-induced oxidative stress (2.5–25 mJ/cm²) (Black et al., 2008a) in either undifferentiated and differentiated mouse keratinocytes. Whether or not these responses protect the skin from UVB will depend on the levels of expression of functional proteins for these enzymes in the different cell types and their precise roles in regulating oxidative stress and/or repairing cellular damage. Environmental chemicals may be involved in the etiology of breast cancer. Among them, organophosphorous compounds are the most widely used pesticides because of their extensive use in agriculture, medicine and industry. MGST1 was upregulated in the organophosphate pesticide parathion-treated group and downregulated in estradiol-treated group in comparison to control. The combination of parathion and estradiol induced downregulation of MGST1 in MCF-10F human breast epithelial cells over 2.5–5 fold (Calaf & Roy, 2007).

5. MGST1 in apoptosis

Mitochondria are the major intracellular source of ROS and end-products from membrane-lipid peroxidation caused by ROS are highly toxic, therefore mitochondrial MGST1 is essential to protect mitochondria from oxidative damage through its activities to catalyze GSH conjugation and GSH-mediated peroxide reduction. In addition, mitochondrial MGST1 may contribute to the regulation of mitochondrial permeability transition (MPT) pore and apoptotic cell death (Aniya & Imaizumi, 2011). MGST1 in the inner mitochondrial membrane (IMM) interacts with MPT regulator proteins, cyclophilin D (Cyt-D) in the mitochondrial matrix and adenine nucleotide translocator (ANT) in the IMM. Under chemical or oxidative stress, mitochondrial MGST1 is activated by S-glutathionylation, or by forming a mixed disulfide bond with Cyt-D or ANT, resulting in conformational changes, opening of the MPT pore, mitochondrial swelling, and cytochrome c release. These events can be inhibited by MPT inhibitors, cyclosporine A (CsA) and bongkrekic acid (BKA), which bind to Cyt-D and ANT, respectively. Binding of CsA/BKA to Cyt-D/ANT causes their conformational change followed by an alteration of mitochondrial MGST1 conformation, resulting in decreased MGST1 activity (Lee, Shimoji, Hossain, Sunakawa, & Aniya, 2008; Ulziikhishig et al., 2010). Alternatively, oxidative activation of MGST1 in the OMM may allow oligomerization or reaction with other proteins to form a pore for cytochrome c release (Hossain, Ulziikhishig, Lee, Yamamoto, & Aniya, 2009; Lee et al., 2008). Recent studies indicate that dendritic cell factor 1 (DCF1), a transmembrane protein, may play an role in maintaining mitochondrial permeability through regulation of mitochondrial MGST1 expression and localization. DCF1 overexpression causes mitochondrial damage and apoptosis, with pronounced mitochondrial swelling, destruction of cristae, and a significant decline in membrane potential (Chen et al., 2018; Xie et al., 2014).

6. MGST1 in ferroptosis

Ferroptosis is a non-apoptotic form of cell death induced by the build-up of toxic lipid ROS. Ferroptosis occurs by three steps: reduced activity of the cysteine-glutamate antiporter (system Xc−); inhibition of selenocysteine-containing glutathione peroxidase 4 (GPX4); iron-dependent generation of lipid peroxides through Fenton chemistry and the enzymes arachidonate lipoxygenase (ALOX) and NADPH oxidases (NOX) (Talty & Bosenberg, 2022). Extensive studies suggest that ferroptosis plays a pivotal role in tumor suppression and inducing ferroptosis can reverse resistance to common drug-, targeted- and immunotherapies (Chen, Kang, Kroemer, & Tang, 2021; Talty & Bosenberg, 2022; Zhang, Liu, Jin, Chen, & Guo, 2022). MGST1 plays a key role in inhibiting ferroptosis in cancer cells and may be a potential therapeutic target (Fig. 1). There is evidence that MGST1 can bind to ALOX5 and suppress ferroptosis by reducing the generation of lipid peroxides (Kuang, Liu, Xie, Tang, & Kang, 2021). MGST1, as a non-selenium dependent GPX, can catalyze the reduction of lipid peroxides (Mosialou et al., 1995; Mosialou, Ekstrom, Adang, & Morgenstern, 1993) and thus protect cells from ferroptosis. In addition, recently we found a positive correlation between MGST1 expression and melanogenesis. Melanin has a distinct protective effect on lipid peroxidation, as binding of ferrous ions by melanin decreases the yield of hydroxyl radicals (Pilas, Sarna, Kalyanaraman, & Swartz, 1988).

Fig. 1.

Microsomal glutathione transferase 1 (MGST1) plays a key role in inhibiting ferroptosis in cancer cells. Ferroptosis occurs by three steps: reduced activity of the cysteine-glutamate antiporter (system Xc−); inhibition of selenocysteine-containing glutathione peroxidase 4 (GPX4); iron-dependent generation of lipid peroxides through Fenton chemistry and the enzymes arachidonate lipoxygenase (ALOX) and NADPH oxidases. Polyunsaturated-fatty-acid-containing phospholipids (PL-PUFA) in membrane undergo lipid peroxidation, which directly destroys the cellular membrane, thereby causing cell death via ferroptosis. MGST1 or GPX4 reduces lipid peroxides to lipid alcohols by oxidizing glutathione (GSH), thereby protecting cells from ferroptosis. In addition, MGST1 can reduce the generation of lipid peroxides (·OH) by binding to arachidonate 5-lipoxygenase (ALOX5) and promoting the melanin biosynthesis. Binding of melanin with ferrous ions (Fe²⁺) decreases the yield of hydroxyl radical through Fenton chemistry. Ferroptosis plays a pivotal role in tumor suppression and inducing ferroptosis has been demonstrated to reverse resistance of cancer to common drug-, targeted- and immunotherapies. Inhibition of MGST1 and induction of ferroptosis may be a novel strategy for cancer therapy.

Uterine corpus endometrial carcinoma (UCEC) is a common gynecological malignancy closely linked with iron, in which cell oxidative stress-associated ferroptosis is dysregulated. Both mRNA and protein levels of MGST1 were higher in UCEC tissue than in normal, and higher MGST1 was associated with different histological types and advanced grades, a lack of hormone therapy, and poor overall, disease specific and progress free survivals. Ferroptosis can significantly enhance antitumor immunity according to the specific phenotypes and functions of infiltrated immune cells. MGST1, by regulating ferroptosis, may change the tumor microenvironment. The expression level of MGST1 was found to be negatively correlated with NK cells and CD8⁺ T cells and positively correlated with myeloid-derived suppressor cells in UCEC. Such results reveal the association between ferroptosis and tumor-infiltrating immune cells with dysregulated MGST1, which has the potential to be involved as a ferroptosis inducer (Yan, Ye, & Shao, 2022).

In our recent study, we identified MGST1 as highly expressed in dedifferentiated and drug resistant human melanomas and as a specific determinant of metastatic spread and therapeutic sensitivity. Loss of MGST1 in mouse B16 and human MNT-1 melanoma enhanced cellular oxidative stress, and sensitivity to cytotoxic anticancer drugs and ferroptotic cell death. When compared to control, mice bearing Mgst1 KD B16 tumors had more CD8⁺ T cell infiltration with reduced expression of inhibitory receptors and increased cytokine response, large reduction of lung metastases and enhanced survival. Taken together, our data indicate that MGST1 inhibitors may provide a potential therapeutic approach in the management of metastatic melanoma. Therefore, enhancing chemo and immune response and decreasing tumor progression through inhibition of MGST1/induction of ferroptosis may be a novel strategy for cancer therapy.

7. MGST1 in cancer

MGST1 overexpression has been demonstrated in various cancers (Table 1). MGST1 was upregulated in patients with acute myeloid leukemia (AML), in contrast to acute lymphoblastic leukemia (ALL), and thus has significance in classifying differences between AML and ALL (Wang & Gotoh, 2009). MGST1 was upregulated in malignant compared to normal prostate tissues derived from the same surgical specimens (Chaib, Cockrell, Rubin, & Macoska, 2001). MGST1, cathepsin H and syndecan 1 were consistently elevated in murine transgenic lung tumors compared to non-tumor bearing transgenic lung tissues and normal lung tissues surrounding the tumors. Importantly, these 3 proteins were also strongly expressed in human Stage I non-small cell lung cancer, suggesting that they may be linked with early-stage human lung adenocarcinomas (LUAD) and/or squamous cell (LUSC) carcinomas (Linnerth, Sirbovan, & Moorehead, 2005).

Table 1.

Microsomal glutathione transferase 1 (MGST1) and cancer.

Type of cancer	Observation Overexpression
Acute myeloid leukemia	MGST1 was upregulated in patients with acute myeloid leukemia compared to acute lymphoblastic leukemia (Wang & Gotoh, 2009).
Prostate cancer	MGST1 was upregulated in malignant prostate tissues (Chaib et al., 2001).
Lung cancer	MGST1 was elevated in murine transgenic lung tumors and strongly expressed in human stage I non-small cell lung cancer (Linnerth et al., 2005).
Ovarian cancer	MGST1 was overexpressed in advanced-stage serous ovarian carcinoma (Hetland et al., 2012).
	Undetectable
Neuroblastoma	MGST1 was undetectable in either human neuroblastoma cell lines or primary tumor tissues (Kelner et al., 2014).
	Polymorphisms and cancer susceptibility
Colorectal cancer	Among Han Chinese, the carriers of combined genotypes of 102 G > A/16416 G > A (GG/GG) showed significantly increased colorectal cancer risk, and the GG haplotype with two risk alleles was associated with significantly increased risk compared with the other haplotypes (Zhang et al., 2007).
	rs7300446, rs12426370, and rs10846370 are the top three ranked SNPs (single nucleotide polymorphism) association with overall survival in metastatic colorectal cancer patients (Innocenti et al., 2021).
	Regular use of aspirin and other non-steroidal anti-inflammatory drugs was associated with a lower risk of colorectal cancer among individuals with rs2965667-TT genotype, but a higher risk among those with the much less common (4%) TA or AA genotypes (Nan et al., 2015).
Ovarian cancer	rs6488840 was significantly associated with reduced serous epithelial ovarian cancer risk in women of African-American ancestry, of borderline significance in women of whit e-European, but not Asian ancestry (Chornokur et al., 2015).
	Cancer biomarker
Lung cancer	There were increased prevalence of lung cancer development among individuals who expressed MGST1 at threshold levels, suggesting MGST1 as a possible risk biomarker (Blomquist et al., 2009).
Cancer predisposition	MGST1 was downregulated in Mitchell–Riley syndrome patients which may be related to cancer predisposition (Calcaterra et al., 2021).
Bladder cancer	MGST1 was one of the genes induced by BCG (Bacillus Calmette-Guérin) immunotherapy and may be a biomarker to evaluate outcomes in bladder cancer patients receiving this therapy (Rahmat et al., 2018).
B-cell lymphomas	MGST1 was proposed to be a host-specific marker of EBV (Epstein-Barr virus) latency III, providing a predictive power in screening these B-cell lymphomas for possible efficacy of T-cell therapies and chemotherapeutics (Messinger et al., 2019).
	Cancer metastases
Colorectal cancer	MGST1 was upregulated in human metastatic colorectal cancer cell line SW620 (Chaib et al., 2001).
Melanoma	MGST1 was upregulated in highly metastatic human melanoma M4Be-Tw12 clone (Bertucci et al., 2007).
	MGST1 was downregulated in catalase overexpressed human metastatic A375-G10 melanoma clone (Bracalente et al., 2016).
	Cancer prognosis and survival
Bone cancer	The expression of MGST1 was found to predict Ewing’s sarcoma and osteosarcoma prognosis, with low expression correlating with a better event-free and overall survival (Scotlandi et al., 2009; Selvarajah et al., 2009).
Hepatocellular carcinoma	MGST1 had a negative impact on the prognosis in hepatocellular carcinoma (Liang et al., 2021).
Lung cancer	Positive expression of MGST1 in lung adenocarcinoma was correlated with the AJCC clinical stage. Higher MGST1 expression was related to lower overall survival rates, and negative MGST1 expression correlated with better prognosis (Zeng et al., 2020a).
Gliomas	MGST1 expression was high in glioma tissues and higher MGST1 in patients tended to be associated with lower survival rates. miR-379–5p has been shown to bind MGST1 and negatively regulate its expression. miR-379–5p was downregulated in glioma tissues, as well as glioma cell lines, and its expression was inversely correlated with viability, migration, and invasion (Yang et al., 2021).
Melanoma	MGST1 was a risk factor gene for overall survival and could be used in a formula to predict individual melanoma prognosis in high and low-risk groups (Zeng et al., 2020b).
Pancreatic ductal adenocarcinoma	MGST1 was upregulated in pancreatic cancer stem cells and was not only positively correlated with the stemness signature, but also associated with reduced disease-free survival in pancreatic ductal adenocarcinoma patients (Jagust et al., 2020).
	Chemo-radiation resistance
Ewing’s sarcoma	The expression of MGST1 was associated with chemoresistance to doxorubicin (Scotlandi et al., 2009).
Pancreatic ductal adenocarcinoma	Higher expression of MGST1 was correlated with gemcitabine resistance (Bai et al., 2007).
Cervical carcinoma	MGST1 was upregulated in human SiHa cervical carcinoma cisplatin resistance cells, and suppression of MGST1 gene expression by pentoxifylline could induce sensitization of cisplatin resistant cells to apoptosis (Bravo-Cuellar et al., 2020).
HER2-negative breast cancer	Systematic down-regulation of long non-coding RNA (IncRNA), especially lnc-MGST1–2, in residual cancer tissues, when compared to paired to remittent tissues of HER2-negative breast cancer after neoadjuvant chemotherapy, contributed to the chemo-resistance process (Ouyang et al., 2018).
Lung cancer	MGST1, MGST3 and GSTO1 were upregulated in human lung adenocarcinoma cisplatin-resistant cell lines and tumor tissues, implying that the network of lncRNA MSTRG51053.2-miR-432–5p-MGST3 may contribute to cisplatin resistance (Zhang et al., 2020).
Mantle cell lymphoma	Bendamustine hydrochloride resistance was mediated by overexpression of ABCB1 and MGST1, and was accompanied by multidrug resistance doxorubicin, mafosfamide, melphalan, vincristine and ibrutinib (Takimoto-Shimomura et al., 2018).
Tumor cell lines	MGST1 expression contributed to the resistance of tumor cells (55 cell lines of the NCI panel) to artesunate and to the low toxicity of artesunate in normal organs (Efferth & Volm, 2005).
Breast cancer	Heterologous overexpression of MGST1 in MCF7 breast cancer cells was shown to protect against cytostatic drugs, e.g. cisplatin, melphalan and chlorambucil (Johansson et al., 2007; Zhang & Lou, 2003; Zhang et al., 2004; Zhang et al., 2005). Prodrugs that are derivatives of existing anticancer drugs and releasable by MGST1 could overcome MGST1 mediated resistance (Johansson et al., 2011; van Gisbergen et al., 2016).
Cervix and head and neck cancers	MGST1 and TFPI were the only genes differentially expressed between low and high SF2 (survival fraction following a single 2 Gy dose of radiation) groups in both cervix and head and neck cancers, and may be useful as common gene signatures to reflect the radiosensitivity of these tumors (Hall et al., 2014).

MGST1 was overexpressed in advanced-stage serous ovarian carcinoma (OC) compared to normal ovarian tissue, where it was undetectable in most samples. Solid tumors had significantly higher MGST1 expression than effusions. Moreover, MGST1 expression in solid tumors obtained from patients with ascites at diagnosis was higher than in patients with no ascites. However, MGST1 levels were unrelated to survival or chemotherapy resistance in OC. It is therefore possible that despite ubiquitous expression in advanced OC, other antioxidant systems are stronger modulators of OC biology. Alternatively, it may be that comparison of early- and advanced-stage OC will identify a role in tumor progression and survival in this cancer (Hetland et al., 2012).

Despite the ubiquitous nature of MGST1 in human tissue and other cell lines, neither human neuroblastoma (NB) cell lines (SK-N-SH and IMR-32) nor NB primary tumor tissues, displayed MGST1 mRNA (Kelner et al., 2014). This finding was unanticipated as MGST1 mRNA expression is ubiquitously expressed primarily under the influence of the SP1 transcription factor (Ekstrom, Lyrenas, Jakobsson, Morgenstern, & Kelner, 2003), and SP1 expression is upregulated in NB cell lines. However, it explained the sensitivity of NB cell lines to oxidative stress, including reports of disruption of mitochondrial potential, and suggested therapeutic opportunities to take advantage of MGST1 absence in NB.

MGST1 has been associated with poor cancer prognosis and survival, high metastatic potential, and chemo-radiation resistance. It has been suggested to be a biomarker for cancer risk assessment and predication of treatment efficacy. MGST1 polymorphisms may change the risk of certain types of cancer. See details in the sections below and the observations are also summarized in Table 1.

7.1. MGST1 polymorphisms and cancer susceptibility

MGST1 polymorphisms may contribute to colorectal cancer (CRC) risk among Han Chinese. The carriers of combined genotypes of 102G > A/16416G > A (GG/GG) showed significantly increased CRC risk (OR = 1.682, 95% CI: 1.177–2.404). Consistently, the GG haplotype with two risk alleles was associated with significantly increased risk compared with the other haplotypes with OR = 1.744, 95% CI: 1.309–2.322. These data were used to imply the potential of developing MGST1 as a CRC susceptibility marker (Zhang et al., 2007).

The top three ranked SNPs (single nucleotide polymorphism) association with overall survival in metastatic CRC patients are rs7300446, rs12426370, and rs10846370. They are intergenic between MGST1 and LMO3 (LIM domain only 3) and belong to a haplotype block (r² > 0.85 among them) (Innocenti et al., 2021).

Use of aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) is associated with lower risk of CRC. However, the mechanisms behind this association are not well understood. In the conventional logistic regression analysis, the SNP rs2965667 at chromosome 12p12.3 near the MGST1 gene showed a genome-wide significant interaction with aspirin and/or NSAID use (P for interaction = 4.6 × 10⁻⁹). Compared to non-use, regular use of aspirin and/or NSAIDs was associated with a lower risk of CRC among individuals with rs2965667-TT genotype (OR = 0.66; 95% CI = 0.61–0.70; P = 7.7 ×10⁻³³), but a higher risk among those with the much less common (4%) TA or AA genotypes (OR = 1.89; 95% CI = 1.27–2.81; P = 0.002). Individuals with different genetic backgrounds may obtain differential benefit from aspirin and NSAID and further validation in additional populations may facilitate targeted colorectal cancer prevention strategies (Nan et al., 2015).

Inherited variation in cellular genes contributes to OC risk. MGST1 rs6488840 was significantly associated with reduced serous epithelial OC risk in women of African-American ancestry (OR = 0.55; P = 0.0035). This SNP was of borderline significance in women of white-European (OR = 0.95; P = 0.042), but not Asian (P > 0.05) ancestry (Chornokur et al., 2015).

7.2. MGST1 as a cancer biomarker for risk assessment and predication of treatment efficacy

There were increased prevalence of lung cancer development among individuals who expressed MGST1 at threshold levels that were either very high or very low in normal airway epithelium cells, suggesting MGST1 as a possible risk biomarker for developing lung cancer (Blomquist et al., 2009).

Mitchell–Riley syndrome (MRS) is an autosomal recessive disorder caused by mutations in the RFX6 gene in which a combination of neonatal diabetes mellitus and congenital gastrointestinal defects occur. The analysis of differentially expressed genes revealed that MGST1 is downregulated in MRS patients which may be related to severe diabetic condition, multi-organ impairment or cancer predisposition (Calcaterra et al., 2021).

BCG (Bacillus Calmette-Guérin) immunotherapy is a standard therapy for non-muscle invasive bladder cancer. To model clinical therapy, human bladder cancer cell lines were incubated with BCG (live or lyophilized BCG Connaught) for 2 h. MGST1 is one of the genes induced by BCG membrane interactions and/or soluble factors, and may be a candidate biomarker to evaluate outcomes in patients with bladder cancer receiving BCG immunotherapy (Rahmat, Esuvaranathan, & Mahendran, 2018).

Epstein-Barr virus (EBV) is a ubiquitous herpes virus prevalent in B-cell lymphomas of immune-suppressed individuals. MGST1 was recently proposed to be a host-specific marker of EBV latency III as its mRNA levels significantly increased from latency IIb to latency III, providing a predictive power in screening these tumors for possible efficacy of T-cell therapies and chemotherapeutics (Messinger, Dai, Stanland, Price, & Luftig, 2019).

7.3. MGST1 and cancer metastatic potential

MGST1 was one of the most differentially upregulated genes in human metastatic CRC cell lines SW620 compared to its non-metastatic counterpart SW480, consistent with the more aggressive phenotype of this cell line (Chaib et al., 2001).

MGST1 was also found to be the second most upregulated gene in a strongly metastatic human melanoma clone Tw12, with log2 ratio over 7 between expression level in Tw12 vs the parental M4Be cell lines (Bertucci et al., 2007). However, in a different study, melanotic A7 and metastatic G10 colonies were obtained from parental human A375 melanoma cells after catalase overexpression with pcDNA3-catalase transfection. MGST1 was upregulated in melanotic A7, whereas it downregulated in metastatic G10 vs controls (Bracalente et al., 2016). The exact role of MGST1 in melanoma metastases, whether it is causative, or only a consequence of the phenotype, remains to be explored.

7.4. MGST1 and cancer prognosis and survival

The expression of MGST1 was found to predict Ewing’s sarcoma prognosis, with low expression correlating with a better event-free and overall survival, and to be associated with chemoresistance to doxorubicin, which is one of the most important drugs in the treatment of this disease (Scotlandi et al., 2009). MGST1 was highly expressed in poor survivors of osteosarcoma and associated with the overall survival in both human and canine diseases (Selvarajah et al., 2009).

Hepatocellular carcinoma (HCC) is a malignant tumor with a high cancer-related mortality. The PPP2CA gene encodes the α subtype of the protein phosphatase 2A (PP2A) catalytic subunit and regulates PP2A activity by selecting the PP2A regulatory subunit. PPP2CA is highly expressed in HCC tissues and is significantly related to gender and tumor-node-metastasis staging of patients. High PPP2CA expression is also associated with poor prognosis and worse overall/recurrence-free survival. Through the protein-protein interaction network, MGST1 was identified as one of the top 9 most highly connected hub genes. High expression of the MGST1 has a negative impact on the prognosis of HCC indicating that the PPP2CA/MGST1 axis may have potential value as a target for new drug discovery (Liang et al., 2021).

MGST1 mRNA levels were upregulated in human LUAD tissues compared to normal lung. MGST1 protein expression was elevated in human LUAD tissues compared with paired non-tumor tissues. Positive expression of MGST1 in LUAD was correlated with the AJCC clinical stage. Higher MGST1 expression in LUAD was related to lower overall survival rates, and negative MGST1 expression correlated with better prognosis. MGST1 expression was significantly increased in a panel of LUAD cell lines, compared with the normal lung bronchial epithelial cell lines. Knockdown of MGST1 in A549 and PC-9 cells inhibits proliferation, increases the apoptosis rates in vitro and reduces LUAD growth in vivo, probably by inactivating components of the AKT/GSK-3β pathway. Further work will be needed to investigate the significance of MGST1 in AKT/GSK-3β activation (Zeng et al., 2020a).

Gliomas are the most common malignant brain tumor, possessing unlimited proliferation and strong invasion capabilities, and are mostly insensitive to radiotherapy and chemotherapy. The average survival time of patients is around 14 months. MGST1 expression was high in glioma tissues and higher MGST1 in patients tended to be associated with lower survival rates. In glioma cell cultures, MGST1 expression was positively correlated with viability, migration, and invasion. miR-379–5p has been shown to bind MGST1 and negatively regulate its expression. In comparison with the human normal tissues and astrocytes, miR-379–5p was downregulated in glioma tissues, as well as glioma cell lines, and its expression was inversely correlated with viability, migration, and invasion. Induced MGST1 expression could restore the acquired phenotype of miR-379–5p upregulation, emphasizing its role in the miR-379–5p/MGST1 axis in glioma (Yang, Xia, Ye, Jing, & Wu, 2021).

Because melanoma has high levels of invasiveness, metastasis, and chemotherapy resistance patient prognosis is frequently poor. Metabolic reprogramming is also a characteristic of melanoma, but the prognostic value of metabolism-related genes (MRGs) remains unclear. Eighteen potential prognostic MRGs were identified using the Cancer Genome Atlas and MGST1 were identified as a primary candidate. MGST1 was considered as a risk factor gene for overall survival and to some extent, it could be used in a formula to predict individual melanoma prognosis in high and low-risk groups (Zeng et al., 2020b).

Like normal stem cells, cellular metabolism is highly regulated in cancer stem cells (CSCs) and governs essential aspects of their functionality. A reduced intracellular redox state with low ROS levels is essential for self-renewal. Pancreatic CSCs are dependent on mitochondrial oxidative phosphorylation for full stemness and tumorigenicity, indicating the powerful antioxidant networks in place and making the redox state of mitochondria a relevant target for CSC elimination. 3 of the upregulated genes (MGST1, GPX8, GCCT) in pancreatic CSCs were not only positively correlated with the stemness signature, but also associated with reduced disease-free survival in pancreatic ductal adenocarcinoma (PDAC) patients [2.2–2.5 times increased risk of recurrence; P = 0.0054, 0.03 and 0.0054, respectively], suggesting a critical role for this pathway in pancreatic cancer progression (Jagust, Alcala, Sainz, Heeschen, & Sancho, 2020). Higher expression of MGST1 was also correlated with gemcitabine resistance in PDAC patients (Bai, Sata, & Nagai, 2007).

7.5. MGST1 and chemo-radiation resistance

Cisplatin is the most effective drug against cervical cancer in neoadjuvant and salvage therapy, but its administration is hindered by the occurrence of resistance. MGST1 was upregulated in human SiHa cervical carcinoma cisplatin resistant cells. Pentoxifylline downregulated the expression of the MGST1 gene, induced sensitization of cisplatin resistant cells to apoptosis with increased caspase-9 and -3 activities, reduced NFkB/p65 phosphorylation, and increased PARP-1 cleavage (Bravo-Cuellar et al., 2020).

Long non-coding RNAs (lncRNAs) are defined as RNA genes longer than 200 bp with no coding potential. Recent studies have shown that lncRNA gene silencing is involved in chromatin modification, transcriptional activation, and many other important biological processes. Systematic down-regulated lncRNAs in residual cancer tissues, when compared to paired remittent tissues of HER2-negative breast cancer after neoadjuvant chemotherapy, contribute to the chemo-resistance process. RT-PCR confirmed that two down-regulated lncRNAs (lnc-MGST1–2 and lnc-BTG2–2), consistent with the microarray analysis, showed a significant difference between residual cancer and remittent tissues. Further studies would be needed to confirm the link between lnc-MGST1–2 and clinical outcomes, as well as the use of lnc-MGST1–2 as a potential diagnostics and/or therapeutic targets for chemo-resistant HER2-negative breast cancer (Ouyang et al., 2018).

Expression levels of MGST1, MGST3 and GSTO1 were increased in the human LUAD cisplatin-resistant cell line when compared to wild type. Additionally, these genes were upregulated among cisplatin-resistant tumor tissues, implying that the network of lncRNA MSTRG51053.2-miR-432–5p-MGST3 may contribute to cisplatin resistance. In this regard, MGST1, MGST3 and GSTO1 may serve as cisplatin drug targets to reverse the resistance in non-small cell lung cancer (Zhang et al., 2020).

Mantle cell lymphoma is one subtype of non-Hodgkin’s lymphoma. Bendamustine hydrochloride (BH) is a reasonably new drug used in its treatment, however little is known about the mechanisms of resistance to BH. A BH-resistant mantle cell lymphoma subline was generated, and gene expression profiling revealed upregulation of 312 genes, including ABCB1 encoding P-glycoprotein (P-gp), and MGST1. Addition of either a P-gp inhibitor or a GST inhibitor, at least partly, restored sensitivity to BH in resistant cells, which showed cross-resistance to various drugs including doxorubicin, mafosfamide, melphalan, vincristine and ibrutinib. Thus, BH resistance is mediated by overlapping mechanisms with overexpression of ABCB1 and MGST1, and is potentially accompanied by multidrug resistance in mantle cell lymphoma (Takimoto-Shimomura et al., 2018).

In addition, GSH-related enzymes were found to contribute to the resistance of tumor cells (55 cell lines of the NCI panel) to artesunate and to the low toxicity of artesunate in normal organs. A tendency for correlation between mRNA expression of MGST1 and IC50 values for artesunate was observed (Efferth & Volm, 2005). MGST1, heterologously expressed in MCF7 breast cancer cells, was shown to protect against oxidative stress (Johansson, Jarvliden, Gogvadze, & Morgenstern, 2010; Siritantikorn et al., 2007) and several cytostatic drugs including known/predicted substrates as well as non-substrates of the enzyme (Johansson et al., 2007; Zhang & Lou, 2003; Zhang, Ye, & Lou, 2004; Zhang, Ye, & Lou, 2005). Prodrugs that are derivatives of existing anticancer drugs and releasable by MGST1 have also been designed to overcome MGST1 mediated resistance (Johansson et al., 2011; van Gisbergen et al., 2016).

Radiotherapy plays an integral part in the management of cervical cancer and head and neck squamous cell carcinoma and measurements of radiosensitivity have been shown to correlate with clinical response. MGST1 and TFPI were the only genes differentially expressed between low and high SF2 (survival fraction following a single 2 Gy dose of radiation) groups in both cervix and head and neck cancers, and may be useful as common gene signatures to reflect the radiosensitivity of these tumors (Hall et al., 2014).

8. Summary

MGST1 has a well-established role in protection from reactive electrophilic intermediates and oxidative stress. Emerging data show that under chemical or oxidative stress, mitochondrial MGST1 may contribute to apoptotic cell death. Distinct from apoptosis, ferroptosis is an intracellular iron-dependent form of cell death induced by the build-up of lipid reactive oxygen species and plays a role in tumor suppression and reversing resistance to common chemo- and immunotherapies. MGST1 inhibits ferroptosis in cancer cells through protein-protein interactions with arachidonate 5-lipoxygenase, or its lipid peroxidase activities. MGST1 is overexpressed in various cancers and may be a therapeutic target since it is associated with poor prognosis/survival, high metastatic potential, and chemo-radiation resistance. It may also be a biomarker for cancer risk assessment and prediction of treatment efficacy. In addition, MGST1 polymorphisms may change individual risk to certain cancers. Inhibition of MGST1 and induction of ferroptosis may be a novel strategy for cancer therapy.