Glutathione S-transferase Omega Research Articles

GSTO1 aggravates EGFR-TKIs resistance and tumor metastasis via deglutathionylation of NPM1 in lung adenocarcinoma.

Despite significantly improved clinical outcomes in EGFR-mutant lung adenocarcinoma, all patients develop acquired resistance and malignancy on the treatment of EGFR tyrosine kinase inhibitors (EGFR-TKIs). Understanding the resistance mechanisms is crucial to uncover novel therapeutic targets to improve the efficacy of EGFR-TKI treatment. Here, integrated analysis using RNA-Seq and shRNAs metabolic screening reveals glutathione S-transferase omega 1 (GSTO1) as one of the key metabolic enzymes that is required for EGFR-TKIs resistance in lung adenocarcinoma cells. Aberrant upregulation of GSTO1 confers EGFR-TKIs resistance and tumor metastasis in vitro and in vivo dependent on its active-site cysteine 32 (C32). Pharmacological inhibition or knockdown of GSTO1 restores sensitivity to EGFR-TKIs and synergistically enhances tumoricidal effects. Importantly, nucleophosmin 1 (NPM1) cysteine 104 is deglutathionylated by GSTO1 through its active C32 site, which leads to activation of the AKT/NF-κB signaling pathway. In addition, clinical data illustrates that GSTO1 level is positively correlated with NPM1 level, NF-κB-mediated transcriptions and progression of human lung adenocarcinoma. Overall, our study highlights a novel mechanism of GSTO1 mediating EGFR-TKIs resistance and malignant progression via protein deglutathionylation, and GSTO1/NPM1/AKT/NF-κB axis as a potential therapeutic vulnerability in lung adenocarcinoma.

Glutathione S-transferase omega class 1 (GSTO1)-associated large extracellular vesicles are involved in tumor-associated macrophage-mediated cisplatin resistance in bladder cancer.

Bladder cancer poses a significant challenge to chemotherapy due to its resistance to cisplatin, especially at advanced stages. Understanding the mechanisms behind cisplatin resistance is crucial for improving cancer therapy. The enzyme glutathione S-transferase omega class 1 (GSTO1) is known to be involved in cisplatin resistance in colon cancer. This study focused on its role in cisplatin resistance in bladder cancer. Our analysis of protein expression in bladder cancer cells stimulated by secretions from tumor-associated macrophages (TAMs) showed a significant increase in GSTO1. This prompted further investigation into the role of GSTO1 in bladder cancer. We found a strong correlation between GSTO1 expression and cisplatin resistance. Mechanistically, GSTO1 triggered the release of large extracellular vesicles (EVs) that promoted cisplatin efflux, thereby reducing cisplatin-DNA adduct formation and enhancing cisplatin resistance. Inhibition of EV release effectively counteracted the cisplatin resistance associated with GSTO1. In conclusion, GSTO1-mediated EV release may contribute to cisplatin resistance caused by TAMs in bladder cancer. Strategies to target GSTO1 could potentially improve the efficacy of cisplatin in treating bladder cancer.

Regulation of Glutathione S-Transferase Omega 1 Mediated by Cysteine Residues Sensing the Redox Environment.

Glutathione S-transferase omega 1 (GstO1) catalyzes deglutathionylation and plays an important role in the protein glutathionylation cycle in cells. GstO1 contains four conserved cysteine residues (C32, C90, C191, C236) found to be mutated in patients with associated diseases. In this study, we investigated the effects of cysteine mutations on the structure and function of GstO1 under different redox conditions. Wild-type GstO1 (WT) was highly sensitive to hydrogen peroxide (H2O2), which caused precipitation and denaturation at a physiological temperature. However, glutathione efficiently inhibited the H2O2-induced denaturation of GstO1. Cysteine mutants C32A and C236A exhibited redox-dependent stabilities and enzyme activities significantly different from those of WT. These results indicate that C32 and C236 play critical roles in GstO1 regulation by sensing redox environments and explain the pathological effect of cysteine mutations found in patients with associated diseases.

The Role of Glutathione Transferase Omega-Class Variant Alleles in Individual Susceptibility to Ovarian Cancer.

The tumor microenvironment is affected by reactive oxygen species and has been suggested to have an important role in ovarian cancer (OC) tumorigenesis. The role of glutathione transferases (GSTs) in the maintenance of redox balance is considered as an important contributing factor in cancer, including OC. Furthermore, GSTs are mostly encoded by highly polymorphic genes, which further highlights their potential role in OC, known to originate from accumulated genetic changes. Since the potential relevance of genetic variations in omega-class GSTs (GSTO1 and GSTO2), with somewhat different activities such as thioltransferase and dehydroascorbate reductase activity, has not been clarified as yet in terms of susceptibility to OC, we aimed to investigate whether the presence of different GSTO1 and GSTO2 genetic variants, individually or combined, might represent determinants of risk for OC development. Genotyping was performed in 110 OC patients and 129 matched controls using a PCR-based assay for genotyping single nucleotide polymorphisms. The results of our study show that homozygous carriers of the GSTO2 variant G allele are at an increased risk of OC development in comparison to the carriers of the referent genotype (OR1 = 2.16, 95% CI: 0.88-5.26, p = 0.08; OR2 = 2.49, 95% CI: 0.93-6.61, p = 0.06). Furthermore, individuals with GST omega haplotype H2, meaning the concomitant presence of the GSTO1*A and GSTO2*G alleles, are more susceptible to OC development, while carriers of the H4 (*A*A) haplotype exhibited lower risk of OC when crude and adjusted haplotype analysis was performed (OR1 = 0.29; 95% CI: 0.12-0.70; p = 0.007 and OR2 = 0.27; 95% CI: 0.11-0.67; p = 0.0054). Overall, our results suggest that GSTO locus variants may confer OC risk.

Abstract 2964: Glutathione S-transferase omega 2 (GSTO2) suppresses the acquisition of the senescent tumor cells phenotype in lung adenocarcinoma

Abstract Cellular senescence is a phenomenon characterized by the cell cycle arrest and the induction of inflammatory responses, and is known to contribute to the development and progression of various diseases. In cancerous tissues, some cancer cells acquire the senescent traits, which are referred to as senescent tumor cells (STCs) and contribute to tumor invasion and metastasis. We have previously demonstrated that the expression of glutathione S-transferase omega 2 (GSTO2), which regulates the glutathione redox balance, is exclusively expressed in cells with regenerative ability such as airway basal cells, Clara cells, and type II alveolar cells in the lungs. Contrastingly, the expression of GSTO2 was completely lost in squamous cell carcinoma of the lung (Cancer Science, 2022); however, we found that some of lung adenocarcinoma expressed GSTO2. In this study, we aimed to clarify the significance of GSTO2 in lung adenocarcinoma, especially in the regulation of senescence and STCs. To examine whether GSTO2 expression affects senescence marker expression, we prepared GSTO2-transfected and mock-transfected lung adenocarcinoma cell line PC-9. Forced GSTO2 expression resulted in significantly reduced expression of senescence markers, such as p21, p53, and p16. We performed double staining for GSTO2 with p16 and confirmed the inverse correlation between them in lung adenocarcinoma tissues, where GSTO2-positive cells tended to express lower p16 level compared with GSTO2-negative cells. Furthermore, GSTO2-transfected PC-9 cells showed lower expression of senescence-associated secretory phenotype factors, such as IL-6, TNFα, and IL-1β than mock-transfected cells. These results suggest that GSTO2 suppresses senescence in lung adenocarcinoma cells. We next examined whether GSTO2 expression affects tumor cell growth because senescence is involved in the cell cycle arrest. Expectedly, MTT assay revealed that GSTO2 overexpression in PC-9 cells significantly promoted cell growth compared with the mock transfected cells. Contrastingly, the scratching assay showed that the cell migration of GSTO2-transfected PC-9 cells was significantly delayed compared with that of the mock-transfected cells. In conclusion, we demonstrated that GSTO2 regulates senescence, resulting in the suppression of cell migration. The expression of GSTO2 in lung adenocarcinoma may contribute to the inhibition of the emergence of STCs, being a cause of invasion and resistance to radiotherapy and chemotherapy. Citation Format: Hiroto Hatano, Ryusuke Sumiya, Teruki Hagiwara, Hiromu Suzuki, Kazuhiko Yamada, Norihiro Kokudo, Yuki Kawamura. Glutathione S-transferase omega 2 (GSTO2) suppresses the acquisition of the senescent tumor cells phenotype in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2964.

Abstract 5573: Loss of glutathione S-transferase omega 2 promotes immunosuppressive tumor microenvironment by the induction of PD-L1 expression in lung adenocarcinoma

Abstract Glutathione S-transferase omega 2 (GSTO2) is known as one of important regulators for the glutathione redox balance. We recently reported that GSTO2 is exclusively expressed in airway basal cells, Clara cells, and type II alveolar cells, which have self-renewal capacity in the lungs; however, its expression was completely lost in lung squamous cell carcinoma (LSCC) (Cancer Science, 2022). In this study, we examined the expression of GSTO2 in lung adenocarcinoma (LAC), another type of lung cancer, and analyzed the relationship between GSTO2 expression and clinicopathological features. We enrolled 139 patients who were diagnosed with LAC and underwent surgery, and immunohistochemically evaluated the expression of GSTO2 using formalin-fixed, paraffin-embedded sections of surgical specimens. Fifty eight of 139 lung adenocarcinoma samples exhibited GSTO2 expression, while remaining 81 cases did not. The expression of GSTO2 in LAC samples was significantly associated with never or light (Brinkman Index < 400) smoking history (P=0.0027) and histological subtypes (lepidic 77%; acinar 44%; papillary 22%; micropapillary 16.7%; solid 9.6%; P<0.0001). There were no significant differences between the groups with respect to pN, pM, EGFR mutation, ALK fusion, and ROS-1 fusion. However, GSTO2 expression in LAC significantly associated with pT1 (P<0.0001), early stage (P<0.0001), and absence of PD-L1 expression (P=0.0027). Moreover, patients with GSTO2-negative LAC had a significantly lower disease-free survival rate than those with GSTO2-positve LAC (P=0.028). To examine whether GSTO2 expression affects PD-L1 expression, we prepared GSTO2-transfected and mock-transfected lung adenocarcinoma cell lines (A549 and PC-9). In both cell lines, mRNA level of PD-L1 was significantly reduced in GSTO2-transfectants. Because Jang RH et al reported that vimentin, reflecting to acquire mesenchymal traits in epithelial-mesenchymal transition (EMT), regulates the PD-L1 expression, we next examined whether GSTO2 overexpression affects the expression of EMT related molecules. There was no difference in a mRNA level of E-cadherin, while the mRNA expression of vimentin and SLUG, a transcriptional inducer of EMT, were significantly suppressed in GSTO2-transfected PC-9. In both cell lines, protein expression of vimentin was also significantly reduced in GSTO2-transfectants. The present study shed light on a novel function of GSTO2 as a PD-L1 regulator, which may contribute to prevention of tumor immune escape. Citation Format: Ryusuke Sumiya, Hiroto Hatano, Teruki Hagiwara, Satoshi Nagasaka, Kazuhiko Yamada, Norihiro Kokudo, Hiromu Suzuki, Yuki I. Kawamura. Loss of glutathione S-transferase omega 2 promotes immunosuppressive tumor microenvironment by the induction of PD-L1 expression in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5573.

Rattus norvegicus Glutathione Transferase Omega 1 Localization in Oral Tissues and Interactions with Food Phytochemicals.

Glutathione transferases are xenobiotic-metabolizing enzymes with both glutathione-conjugation and ligandin roles. GSTs are present in chemosensory tissues and fluids of the nasal/oral cavities where they protect tissues from exogenous compounds, including food molecules. In the present study, we explored the presence of the omega-class glutathione transferase (GSTO1) in the rat oral cavity. Using immunohistochemistry, GSTO1 expression was found in taste bud cells of the tongue epithelium and buccal cells of the oral epithelium. Buccal and lingual extracts exhibited thiol-transferase activity (4.9 ± 0.1 and 1.8 ± 0.1 μM/s/mg, respectively). A slight reduction from 4.9 ± 0.1 to 4.2 ± 0.1 μM/s/mg (p < 0.05; Student's t test) was observed in the buccal extract with 100 μM GSTO1-IN-1, a specific inhibitor of GSTO1. RnGSTO1 exhibited the usual activities of omega GSTs, i.e., thiol-transferase (catalytic efficiency of 8.9 × 104 M-1·s-1), and phenacyl-glutathione reductase (catalytic efficiency of 8.9 × 105 M-1·s-1) activities, similar to human GSTO1. RnGSTO1 interacts with food phytochemicals, including bitter compounds such as luteolin (Ki = 3.3 ± 1.9 μM). Crystal structure analysis suggests that luteolin most probably binds to RnGSTO1 ligandin site. Our results suggest that GSTO1 could interact with food phytochemicals in the oral cavity.

Pivotal Role of GSTO2 in Ferroptotic Neuronal Injury After Intracerebral Hemorrhage

Previous research has found that an adaptive response to ferroptosis involving glutathione peroxidase 4 (GPX4) is triggered after intracerebral hemorrhage. However, little is known about the mechanisms underlying adaptive responses to ferroptosis. To explore the mechanisms underlying adaptive responses to ferroptosis after intracerebral hemorrhage, we used hemin-treated HT22 cells to mimic brain injury after hemorrhagic stroke in vitro to evaluate the antioxidant enzymes and performed bioinformatics analysis based on the mRNA sequencing data. Further, we determined the expression of GSTO2 in hemin-treated hippocampal neurons and in a mouse model of hippocampus-intracerebral hemorrhage (h-ICH) by using Western blot. After hemin treatment, the antioxidant enzymes GPX4, Nrf2, and glutathione (GSH) were upregulated, suggesting that an adaptive response to ferroptosis was triggered. Furthermore, we performed mRNA sequencing to explore the underlying mechanism, and the results showed that 2234 genes were differentially expressed. Among these, ten genes related to ferroptosis (Acsl1, Ftl1, Gclc, Gclm, Hmox1, Map1lc3b, Slc7a11, Slc40a1, Tfrc, and Slc39a14) were altered after hemin treatment. In addition, analysis of the data retrieved from the GO database for the ten targeted genes showed that 20 items on biological processes, 17 items on cellular components, and 19 items on molecular functions were significantly enriched. Based on the GO data, we performed GSEA and found that the glutathione metabolic process was significantly enriched in the hemin phenotype. Notably, the expression of glutathione S-transferase omega (GSTO2), which is involved in glutathione metabolism, was decreased after hemin treatment, and overexpression of Gsto2 decreased lipid reactive oxygen species level in hemin-exposed HT22 cells. In addition, the expression of GSTO2 was also decreased in a mouse model of hippocampus-intracerebral hemorrhage (h-ICH). The decreased expression of GSTO2 in the glutathione metabolic process may be involved in ferroptotic neuronal injury following hemorrhagic stroke.

Manganese induces oxidative damage in the hippocampus by regulating the expression of oxidative stress-related genes via modulation of H3K18 acetylation.

Prolonged exposure to manganese (Mn) contributes to hippocampal Mn accumulation, which leads to neurodegenerative diseases called manganese poisoning. However, the underlying molecular mechanisms remain unclear and there are no ideal biomarkers. Oxidative stress is the essential mechanisms of Mn-related neurotoxicity. Furthermore, histone acetylation has been identified as being engaged in the onset and development of neurodegenerative diseases. Therefore, the work aims to understand the molecular mechanisms of oxidative damage in the hippocampus due to Mn exposure from the aspect of histone acetylation modification and to assess whether H3K18 acetylation (H3K18ac) modification level in peripheral blood reflect Mn-induced oxidative damage in the hippocampus. Here, we randomly divided 60 male rats into four groups and injected them intraperitoneally with sterile pure water and MnCl2 ⋅4H2 O (5, 10, and 15 mg/kg) for 16 weeks, 5 days a week, once a day. The data confirmed that Mn exposure down-regulated superoxide dismutase activity and glutathione level as well as up-regulated malondialdehyde level in the hippocampus and plasma, and that there was a positive correlation between these indicators in the hippocampus and plasma. Besides, we noted that Mn treatment upregulated H3K18ac modification levels in the hippocampus and peripheral blood and that H3K18ac modification levels correlated with oxidative stress. Further studies demonstrated that Mn treatment decreased the amounts of H3K18ac enrichment in the manganese superoxide dismutase (SOD2) and glutathione transferase omega 1 (GSTO1) gene promoter regions, contributing to oxidative damage in the hippocampus. In short, our results demonstrate that Mn induces oxidative damage in the hippocampus by inhibiting the expression of SOD2 and GSTO1 genes via modulation of H3K18ac. In assessing Mn-induced hippocampal neurotoxicity, oxidative damage in plasma may reflect hippocampal oxidative damage in Mn-exposed groups.

Ectopic pregnancy: search for biomarker in salivary proteome

Ectopic pregnancy (EP) is associated with high maternal morbidity and mortality. Ultrasonography is the only dependable diagnostic tool for confirming an ectopic pregnancy. In view of inadequate early detection methods, women suffer from a high-life risk due to the severity of EP. Early detection of EP using pathological/molecular markers will possibly improve clinical diagnosis and patient management. Salivary proteins contain potential biomarkers for diagnosing and detecting various physiological and/or pathological conditions. Therefore, the present investigation was designed to explore the salivary proteome with special reference to EP. Gel-based protein separation was performed on saliva, followed by identification of proteins using Liquid Chromatography-Tandem Mass Spectrometry (LC–MS/MS). Totally, 326 proteins were identified in the salivary samples, among which 101 were found to be specific for ruptured ectopic pregnancy (EPR). Reactome analysis revealed innate immune system, neutrophil degranulation, cell surface interactions at the vascular wall, and FCERI-mediated NF-kB activation as the major pathways to which the salivary proteins identified during EPR are associated. Glutathione-S-transferase omega-1 (GSTO1) is specific for EPR and has been reported as a candidate biomarker in the serum of EPR patients. Therefore, saliva would be a potential source of diagnostic non-invasive protein biomarker(s) for EP. Intensive investigation on the salivary proteins specific to EP can potentially lead to setting up of a panel of candidate biomarkers and developing a non-invasive protein-based diagnostic kit.

Gbb glutathionylation promotes its proteasome-mediated degradation to inhibit synapse growth.

Glutathionylation is a posttranslational modification involved in various molecular and cellular processes. However, it remains unknown whether and how glutathionylation regulates nervous system development. To identify critical regulators of synapse growth and development, we performed an RNAi screen and found that postsynaptic knockdown of glutathione transferase omega 1 (GstO1) caused significantly more synaptic boutons at the Drosophila neuromuscular junctions. Genetic and biochemical analysis revealed an increased level of glass boat bottom (Gbb), the Drosophila homolog of mammalian bone morphogenetic protein (BMP), in GstO1 mutants. Further experiments showed that GstO1 is a critical regulator of Gbb glutathionylation at cysteines 354 and 420, which promoted its degradation via the proteasome pathway. Moreover, the E3 ligase Ctrip negatively regulated the Gbb protein level by preferentially binding to glutathionylated Gbb. These results unveil a novel regulatory mechanism in which glutathionylation of Gbb facilitates its ubiquitin-mediated degradation. Taken together, our findings shed new light on the crosstalk between glutathionylation and ubiquitination of Gbb in synapse development.

Ficus deltoidea Leaf Alters Oxidative Stress, Protein Homeostasis and Ubiquitin-Proteasome Pathways in Fatty Acid-Induced Cell Line.

Ficus deltoidea (mistletoe fig) is a shrub well known among locals in Malaysia primarily for its treatment of toothaches, colds and wounds. The aim of this study is to determine the potential of leaves, sourced from three different varieties of F. deltoidea, to exhibit antioxidant activity, a reduction of lipid concentration, and protein expression in steatosis-induced liver cell lines. The leaves of three F. deltoidea varieties, namely Ficus deltoidea var. angustifolia, Ficus deltoidea var. trengganuensis and Ficus deltoidea var. kunstleri, were subjected to water extraction. The resulting crude extracts were fractionated using water and ethyl acetate. Palmitic acid was used to induce lipid accumulation (steatosis) in human liver (WRL68) cells, before all the samples were tested for their lipid-reducing activity. Several proteomic approaches were incorporated. The changes in protein expression were determined using 2-dimensional gel electrophoresis separation, whereas identification of our protein spots of interest was carried out via matrix-assisted laser desorption/ionization time-of-flight. Ficus deltoidea var. kunstleri alone demonstrated the ability to reduce lipids at the highest tested concentration (200 µg/mL) and was, therefore, used for subsequent experiments. Treatment with Ficus deltoidea var. kunstleri was found to restore redox status by increasing superoxide dismutase and glutathione peroxidase amounts and decreasing malondialdehyde formation. Six proteins were successfully identified; these were heat shock protein beta-1 (HSPB1), proteasome subunit alpha type 1 (PSMA1), glutathione S-transferase omega 1 (GSTO1), peroxiredoxin-1 (PRDX1), histone H2B (HIST1H2BD) and ubiquitin c-terminal hydrolase L3 (UCHL3). Through bioinformatics analysis, it was found that these proteins were significantly involved in specific pathways such as oxidative stress (PRDX1 and GSTO1), protein homeostasis (HSPB1) and degradation (UCHL3 and PSMA1). F. deltoidea pretreatment was shown to reduce lipid accumulation, thus improving the redox status and protein homeostasis. This suggests the role of F. deltoidea as a preventive mechanism in non-alcohol fatty liver disease.

Inhibition of glutathione S-transferase omega 1-catalyzed protein deglutathionylation suppresses adipocyte differentiation

Inhibition of glutathione S-transferase omega 1-catalyzed protein deglutathionylation suppresses adipocyte differentiation

Abstract 2602: Loss of glutathione S-Transferase omega 2 is associated with PD-L1 expression and poor prognosis in lung adenocarcinoma

Abstract Glutathione S-transferase omega 2 (GSTO2) lacks any appreciable GST activity but presents thioltransferase activity and therefore plays an important role in regulating the glutathione redox balance. In addition, GSTO2 polymorphisms are strongly associated with lung function and the risk of chronic obstructive pulmonary disease. We recently reported that GSTO2 is exclusively expressed in airway basal cells, Clara cells, and type II alveolar cells, which have self-renewal capacity in the lungs. In this study, we examined the expression of GSTO2 in non-small cell lung cancer (NSCLC) and analyzed the relationship between GSTO2 expression and clinicopathological features. We enrolled 233 patients who were diagnosed with NSCLC and underwent surgery, and immunohistochemically evaluated the expression of GSTO2 using formalin-fixed, paraffin-embedded sections of surgical specimens. While all 94 squamous cell carcinoma samples showed no GSTO2 expression, 58 of 139 lung adenocarcinoma (LAC) samples exhibited GSTO2 expression. The expression of GSTO2 in LAC samples was significantly associated with never or light (Brinkman Index &amp;lt; 400) smoking history (P=0.0027) and histological subtypes (lepidic 85/110, 77%; acinar 30/68, 44%; papillary 18/64, 22%; micropapillary 1/6, 16.7%; solid 3/31, 9.6%; P&amp;lt;0.0001). There were no significant differences between the groups with respect to pN, pM, EGFR mutation, ALK fusion, and ROS-1 fusion. However, GSTO2 expression in LAC significantly associated with pT1 (P&amp;lt;0.0001), early stage (P&amp;lt;0.0001), and absence of PD-L1 expression (P=0.0027). Moreover, patients with GSTO2-negative LAC had a significantly lower disease-free survival rate than those with GSTO2-positve LAC (P=0.028). To examine whether GSTO2 expression affects PD-L1 expression, we prepared GSTO2-transfected and mock-transfected NSCLC cell lines (A549, PC-9, and H520). However, there was no difference in PD-L1 transcription between the GSTO2 and mock transfectants. Previous studies have reported that PD-L1 protein expression is induced via the mitogen-activated protein kinase (MAPK) signaling pathways in lung cancer, glioblastoma, and hepatocellular carcinoma. Our prior finding that GSTO2 accelerates the phosphorylation of p38, a key protein of MAPK signaling (Cancer Science, 2022), suggests that GSTO2 may be involved in PD-L1 expression as a post-transcriptional regulator. In summary, our study indicates that GSTO2 loss contributes to LAC progression partially through the induction of PD-L1 expression. Citation Format: Ryusuke Sumiya, Hiroto Hatano, Teruki Hagiwara, Satoshi Nagasaka, Hiromu Suzuki, Kazuhiko Yamada, Norihiro Kokudo, Yuki I. Kawamura. Loss of glutathione S-Transferase omega 2 is associated with PD-L1 expression and poor prognosis in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2602.

The Upregulation of GSTO2 is Associated with Colon Cancer Progression and a Poor Prognosis.

Colorectal cancer is the second-leading cause of cancer-related mortality in the United States. Glutathione S-transferase can affect the development of cancer. Glutathione S-transferase omega 2, a member of the GST family, plays an important role in many tumors. However, the role of Glutathione S-transferase omega 2 in the development of colon cancer remains unclear. Herein, our study aimed to investigate the exact role of Glutathione S-transferase omega 2 in colon cancer. We used RNA sequencing data from The Cancer Genome Atlas and the Genotype-Tissue Expression database to analyze Glutathione S-transferase omega 2 expressions. Then, we explore the protein information of Glutathione S-transferase omega 2 in the Human Protein Atlas, GeneCards, and String database. In addition, western blot and immunohistochemistry were performed to evaluate the protein levels of Glutathione S-transferase omega 2 in colon cancer tissues. We acquire data from the Gene Expression Omnibus and The Cancer Genome Atlas databases. Also, we performed relevant prognostic analyses of these data. In addition, we performed a statistical analysis of the clinical data from The Cancer Genome Atlas database and the expression level of Glutathione S-transferase omega 2. Then, we performed Cox regression analysis and found independent risk factors for prognosis in patients with colon cancer. The Kyoto Encyclopedia of Genes and Genomes and Gene Ontology enrichment analyses were used to explore the potential biological functions of Glutathione S-transferase omega 2. The infiltration of colon cancer-immune cells was evaluated by the CIBERSORT method. RNA silencing was performed using siRNA constructs in HCT-116 and HT-29 cell lines. Cell Counting Kit-8 and EdU assays were performed to determine cell proliferation. Transwell experiments and scratch tests were used to determine cell migration. As for the mRNA and protein expression levels of cells, we used quantitative real-time PCR and western blot to detect them. Our research shows that Glutathione S-transferase omega 2 is overexpressed in colon cancer patients, and this overexpression is associated with a poor prognosis. The high expression of Glutathione S-transferase omega 2 is significantly correlated stage with stage, M, and N classification progression in colon cancer by statistical analysis. Univariate and multivariate Cox regression analyses showed that Glutathione S-transferase omega 2 was an independent risk factor for poor prognosis in colon cancer. In addition, we also found that Glutathione S-transferase omega 2 expression levels can affect the immune microenvironment of colon cancer cells. Gene silencing of Glutathione S-transferase omega 2 in HT-29 and HCT-116 cells significantly inhibited tumor growth and migration. In summary, we found that Glutathione S-transferase omega 2 can be used as a molecular indicator of colon cancer prognosis. In vitro, gene silencing of Glutathione S-transferase omega 2 inhibited colon cancer cells' growth and migration.

Data-Independent Acquisition-Based Serum Proteomic Profiling of Adult Moyamoya Disease Patients Reveals the Potential Pathogenesis of Vascular Changes.

Moyamoya disease (MMD) is a chronic cerebrovascular disease with unknown etiology. The pathogenesis of vascular changes remains unclear. Ischemic and hemorrhagic adult MMD patients and healthy volunteers were enrolled to collect serum for data-independent acquisition (DIA)-based proteomic analysis and ELISA validation. DIA serum proteomic revealed that apolipoprotein C-I (APOC1), apolipoprotein D (APOD), and apolipoprotein A-IV (APOA4) were decreased. The reductases glutathione S-transferase omega-1 (GSTO1) and peptidyl-prolyl cis-trans isomerase A (PPIA) were upregulated, and ADAMTS-like protein 4 (ADAMTSL4) was downregulated in both ischemic and hemorrhagic MMD. Afamin (AFM) and transforming growth factor-beta-induced protein ig-h3 (TGFBI) increased in ischemic patients but decreased in hemorrhagic patients. Serum ELISA results confirmed that APOA4, APOC1, and APOD were decreased compared to controls. Then, we retrospectively analyzed biochemical indexes of 200 MMD patients. A total of 54 enrolled MMD patients showed decreased total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-c). APOA4, APOC1, and APOD were vital factors in the HDL decrease in MMD patients. Lipoprotein dysfunction in MMD patients is involved in MMD. Intimal thickening by enhanced adhesion, middle layer vascular smooth muscle cell migration, and decreased lipid antioxidant function represented by HDL are potential pathogeneses of vascular changes in MMD.

Knockdown of glutathione S-transferase leads to mislocalization and accumulation of cabeza, a drosophila homolog of FUS, in the brain

Glutathione S-transferase omega (GSTO) is an antioxidant enzyme involved in reducing oxidative stress. Recent studies suggest that polymorphic variants of GSTOs affect the onset age and progression of neurodegenerative diseases. Although GSTO activity may affect the development and age dependency of several diseases, the mechanism by which GSTO inactivation in neurons regulates the susceptibility to neurodegenerative diseases is unclear. In the present study, GstO2 knockdown in Drosophila led to increased levels of Cabeza (Caz) protein in neurons in an age-dependent manner. Drosophila Caz is the ortholog of human FUS, which is associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We found that cytoplasmic Caz mislocalization and aggregation in neurons significantly increased after GstO2 knockdown in vivo. Downregulation of GstO2 decreased the solubility of the Caz protein in aging neurons. These findings demonstrate that GSTO is a critical modulator of the development of neurodegenerative diseases by regulating Caz localization and aggregation in the nervous system of Drosophila.

Differential susceptibility to arsenic in glutathione S-transferase omega 2 (GST-O2)-targeted freshwater water flea Daphnia magna mutants

To examine the role of glutathione S-transferase omega class (GST-O2) genes in the biotransformation and detoxification in Daphnia magna, various responses such as in vivo endpoints, arsenic speciation, enzymatic activities, and gene expression pathways related to arsenic metabolism were investigated in wild-type (WT) and GST-O2–mutant-type (MT) fleas produced by CRISPR/Cas9. Sensitivity to arsenic in MT fleas was higher than in WT fleas. Also, the reduction rate of arsenate (AsV) to arsenite (AsIII) in the MT group was significantly lower and led to accumulation of higher arsenic concentrations, resulting in decreased protection against arsenic toxicity. Relative mRNA expression of other GST genes in the GST-O2–targeted MT group generally increased but the enzymatic activity of GST decreased compared with the WT group. Oxidative stress on arsenic exposure was more strongly induced in the MT group compared with the WT group, resulting in a decrease in the ability to defend against toxicity in GST-O2–targeted mutant D. magna. Our results suggest that GST-O2 plays an important role in arsenic biotransformation and detoxification functions in D. magna.

The Role of CD40, CD86, and Glutathione S-Transferase Omega 1 in the Pathogenesis of Chronic Obstructive Pulmonary Disease.

Objective The aim of the study was to explore the relevance of CD40, CD86, and GSTO1 with the pathogenesis of COPD. Methods Patients with acute exacerbation of COPD were contrasted with the healthy and nonsmoking ones and smoking but without COPD ones. The changes of CD40, CD86, and GSTO1 in the peripheral blood, collected from different groups, were detected by flow cytometry and western blotting, respectively. Results Compared with the nonsmoking group and smoking but without the COPD group, the expression of CD40 and CD86 of the patients with COPD increased significantly, but the expression of GSTO1 decreased. CD40 and CD86 were negatively correlated with FEV1%, while GSTO1 was positively correlated with FEV1% and negatively correlated with CD40 and CD86. Conclusion CD40, CD86, and GSTO1 may play a role in the pathogenesis of COPD, and they are related to the severity of COPD and the degree of changes in the lung function.

GLOBAL PROTEOMIC PROFILING REVEALS METABOLIC REPROGRAMMING IN AMELOBLASTOMAS

<h3>Objectives</h3> To compare the proteome of ameloblastomas and dental follicles. <h3>Study Design</h3> We performed liquid chromatography-tandem mass spectrometry in tumor-enriched samples. Protein-protein interaction network, KEGG-enriched pathways, and gene ontology analyses of differentially abundant proteins were performed. To investigate the activation of the antioxidant system, we assessed the immunoexpression of the glutathione-S-transferase omega-1 (GSTO1). We also examined the status of BRAF mutation in the tumor cases. Statistical analysis was performed using Perseus software. <h3>Results</h3> Ameloblastomas were shown to harbor a proteomic profile distinct from that found in dental follicles, with 33 overregulated and 21 downregulated proteins. Overregulated proteins are involved in glucose metabolism and biosynthesis pathways. Most of the downregulated proteins play prominent roles in cellular mitochondrial energy production and oxidoreductase metabolism regulation. BRAF P.V600E was detected in most ameloblastomas. Ameloblastomas showed diffuse and moderate to strong GSTO1 immunoexpression, whereas weak or negative immunoexpression was observed in dental follicles. <h3>Conclusions</h3> We were able to identify alterations in critical metabolic pathways for the first time, which not only contributes to the elucidation of ameloblastoma pathogenesis but also could be potential targets for drug therapy in these tumors.