Loss of GSTO2 contributes to cell growth and mitochondria function via the p38 signaling in lung squamous cell carcinoma

Abstract Glutathione S‐transferase omega 2 (GSTO2) lacks any appreciable GST activity, but it exhibits thioltransferase activity. The significance of GSTO2 in lung function has been reported; however, the precise expression and molecular function of GSTO2 in the lungs remain unclear. In the present study, we found that GSTO2 is expressed in airway basal cells, non–ciliated, columnar Clara cells, and type II alveolar cells, which have self‐renewal capacity in the lungs. Contrastingly, no GSTO2 expression was observed in 94 lung squamous cell carcinoma (LSCC) samples. When human LSCC cell lines were treated with 5‐aza‐2′‐deoxycytidine, a DNA‐methyltransferase inhibitor, GSTO2 transcription was induced, suggesting that aberrant GSTO2 hypermethylation in LSCC is the cause of its downregulation. Forced GSTO2 expression in LSCC cell lines inhibited cell growth and colony formation in vitro. In a subcutaneous xenograft model, GSTO2‐transfected cells formed smaller tumors in nude mice than mock‐transfected cells. Upon intravenous injection into nude mice, the incidence of liver metastasis was lower in mice injected with GSTO2‐transfected cells than in those injected with mock‐transfected cells. In addition, GSTO2 induction suppressed the expression of β‐catenin and the oxygen consumption rate, but it did not affect the extracellular acidification rate. Furthermore, GSTO2‐transfected cells displayed lower mitochondrial membrane potential than mock‐transfected cells. When GSTO2‐transfected cells were treated with a p38 inhibitor, β‐catenin expression and mitochondrial membrane potential were recovered. Our study indicated that the loss of GSTO2 via DNA hypermethylation contributes to the growth and progression of LSCC, probably by modulating cancer metabolism via the p38/β‐catenin signaling pathway.


| INTRODUC TI ON
Non-small cell lung cancer (NSCLC) is the most frequent type of lung cancer. Lung squamous cell carcinoma (LSCC) is a subtype of NSCLC, and its prognosis is poor. 1 The bronchi and bronchiole are covered with a variety of epithelial cell populations, such as ciliated cells, goblet cells, and Clara cells, which constitute a pseudostratified barrier that protects the lungs from infectious agents and inhaled particulates. 2 Airway basal cells have long-term self-renewal capacity and generate ciliated and secretory luminal cells during homeostasis and epithelial repair, and LSCC arises from airway basal cells. 3 Although homeostatic turnover of the bronchiolar epithelium is low compared to that in other self-renewing tissues, such as the intestine and stomach, all regions in the murine airway can be repaired after injury. 4 In the proximal airway, both basal and Clara cells can generate ciliated cells in the mouse bronchiole. 5 LSCC also arose from Clara and type II alveolar cells in a mouse model featuring genetic alterations commonly found in human LSCC, such as overexpression of SOX2 and loss of Cdkn2ab and Pten. 6 Type II alveolar cells are considered the major stem cells of the distal airway and an origin of lung adenocarcinoma, another subtype of NSCLC. Molecular targeted drugs have been developed for driver mutations observed in NSCLC, such as epidermal growth factor receptors (EGFR), anaplastic lymphoma kinase m, and c-ROS oncogene 1 mutations. [7][8][9] These agents are effective against lung adenocarcinoma, but they do not improve the prognosis of patients with LSCC because the aforementioned driver mutations are frequently observed in lung adenocarcinoma but not commonly present in LSCC. 10 For example, activating mutations of EGFR are present in 35% of patients with lung adenocarcinoma compared to less than 4% of patients with LSCC. 11 Therefore, novel anti-cancer agents based on the molecular mechanisms of LSCC carcinogenesis/progression are urgently needed.
Inhalation of toxic pollutants and microorganisms causes lung injury, which increases the risk of lung cancer. Reduced glutathione (GSH), the most abundant antioxidant expressed in the lung, plays an important role in protecting lung epithelial cells from injury and inflammation following a variety of insults. 12 Previous studies suggested that genetic variations of genes involved in the synthesis, modification, and activation of GSH, including glutathione S-transferase (GST) theta 1, GST pi 1, GST mu 1, 13 GST omega 2 (GSTO2), 14 glutamatecysteine ligase catalytic subunit, and glutathione synthetase, 15 contribute to the risk of chronic obstructive pulmonary disease (COPD), which is an independent risk factor for lung cancer. Among them, GSTO2 polymorphism displayed strong associations with the primary lung function parameters forced expiratory volume in 1 second and forced vital capacity in recent genome-wide association (GWA) analyses, suggesting the significance of GSTO2 in pulmonary function. 16 GSTO2 belongs to the GST superfamily omega class, in which two GSTO genes have been identified in humans. In the lungs, GSTO1 is expressed in alveolar macrophages, airway and alveolar epithelium, and extracellular fluids, including sputum supernatant. 17 Conversely, GSTO2 expression in the lungs has never been elucidated under physiological or pathological conditions, such as COPD and lung cancer.
Of interest, GSTO2 lacks any appreciable GST activity despite belonging to the GST family, which detoxifies xenobiotic substrates, such as tobacco smoke via GSH conjugation. In contrast, GSTO2 exhibits thioltransferase activity, which may have a potential role in regulating GSH levels. 18,19 In the glutathione-ascorbate cycle, redox coupling between GSH and dehydroascorbic acid influences the balance between the oxidized (NADP + ) and reduced (NADPH) forms of nicotinamide adenine dinucleotide phosphate, which are converted into adenosine triphosphate (ATP) in the tricarboxylic acid (TCA) cycle to generate cellular energy. Thus, energy generation depends on oxidation and reduction reactions in mitochondria, and NADPH and GSH are indispensable for these oxidation and reduction reactions. 20 Therefore, GSTO2 may have an essential role in modulating energy metabolism through the regulation of cellular redox status.
The present study aimed to clarify the significance of GSTO2 in the lungs. In this study, we detected GSTO2 expression in airway basal cells, Clara cells, and type II alveolar cells, which correspond to stem cells in the lungs, and loss of GSTO2 expression was confirmed in LSCC. Upon restoring GSTO2 expression in LSCC cells, we found that GSTO2 suppresses cell growth both in vitro and in vivo. We also revealed that forced expression of GSTO2 led to inhibition of mitochondrial function, suggesting that GSTO2 plays an important role in the regulation of energy metabolism in the lungs.

| Patients
We enrolled 94 patients who received a diagnosis of LSCC and underwent lobectomy, segmentectomy, or wedge resection between January 2011 and September 2018 at the National Center for Global Health and Medicine (NCGM). This study was approved by the NCGM research ethics committee (3037), and consent was retrospectively obtained from these patients. Formalin-fixed, paraffin-embedded sections of surgical specimens were used for immunohistochemical analyses.

| Immunohistochemical analysis
Paraffin-embedded sections containing formalin-fixed LSCC specimens were deparaffinized and rehydrated. Target Retrieval Solution  and H520 cells were cultured in RPMI1640 medium supplemented with 10% fetal calf serum (FCS), and EBC-1 was cultured in MEM medium supplemented with 10% FCS. In some experiments, the cells were cultured for 18 hours in a 24-well plate at a density of 2 × 10 4 cells/well and then treated with 5-aza-2′-deoxycytidine (5-aza-dC, Sigma-Aldrich) for 96 hours. EBC-1 and H520 cells, which had been authenticated by the supplier using short tandem repeats (STR), were passaged in our laboratory for fewer than 6 months after resuscitation. LK-2 cells were confirmed to be the same cell line registered in the RIKEN BRC Cell Bank via comparisons with STR profiles on 19 July 2021.
The bisulfite-PCR product was purified and cloned into a pCR4-TOPO vector (Invitrogen), and clones for subsequent sequencing were randomly selected.

| Ectopic expression of GSTO2
A GSTO2 expression vector was transfected into LK-2 and H520 cells using Lipofectamine LTX Reagent (Life Technologies) as previously described, 21 and stable transfectants were isolated using a MoFlo XDP Cell Sorter (Beckman Coulter).

| Cell proliferation assays and colony formation assays
Viable LK-2 and H520 transfectants (2 × 10 3 cells/well) were seeded into 96-well plates, and cell proliferation was measured using an MTT assay kit (Nacalai Tesque). For the colony formation assays, LK-2 and H520 (200 cells/well) transfectants were seeded into 24well plates. After incubation for 2 weeks, colonies were stained with crystal violet.

| Xenograft model in athymic mice
Male BALB/c nude (nu/nu) mice were purchased from CLEA Japan
Briefly, LK-2 and H520 transfectants were incubated with JC-1 dye at a concentration of 5 μg/mL for 30 minutes at 37°C. Images of dye-loaded cells were captured using an In Cell Analyzer (Carl Zeiss).

| Statistical analysis
The data were expressed as the mean ± SD, and the results were compared using paired or unpaired Student's t tests. Statistical analyses were performed using the Prism 8 statistical program (GraphPad Software). All tests were two-tailed, and P < .05 denoted statistical significance.

| GSTO2 expression was suppressed by aberrant DNA hypermethylation in lung squamous cell carcinoma
To investigate the protein expression and localization of GSTO2 in normal lung and LSCC specimens, immunohistochemical analyses were performed. In the normal bronchial epithelium, GSTO2 protein was detected in the nuclei of most basal cells ( Figure 1A). Specific GSTO2 expression in airway basal cells was confirmed by double staining for GSTO2 and CK5/6, which is a marker of airway basal cells 22 ( Figure 1B).
In addition, we detected GSTO2 expression in a minor population of non-basal epithelial cells in the bronchi and bronchiole. This was clearly visualized by double staining for GSTO2 and CC16, which is secreted by the non-ciliated Clara cells in the bronchi and bronchiole, 23 illustrating that GSTO2 protein was also expressed in Clara cells ( Figure 1C).
In the normal alveoli, GSTO2 protein was detected in alveolar type II cells ( Figure 1D), which was confirmed by double staining for GSTO2 and SP-A, a lung-specific glycoprotein in pulmonary surfactant that is secreted by alveolar type II cells 24 ( Figure 1E). GSTO2 expression was detected in 80% of normal tissues surrounding the LSCC (Table 1). In contrast, all 94 LSCC specimens examined in this study displayed no GSTO2 expression in an immunohistochemical analysis ( Figure 1F).
LSCC is classified according to the location of the tumor into either a central or a peripheral type, and the clinicopathologic features of LSCC are significantly different between the two types. 25 Our present study enrolled both LSCC types; therefore, loss of GSTO2 occurred in central LSCC and peripheral LSCC. Loss of GSTO2 was also observed in 60% of dysplasia, which is the precancerous lesion of LSCC ( Figure 1G and increased GSTO2 mRNA expression ( Figure 1H). The methylation status of GSTO2 was assessed using bisulfite sequencing. As expected, methylated CpGs were hardly detected in normal tissues, whereas it was evident that the promoter region of the GSTO2 was frequently hypermethylated in LSCC tissues ( Figure 1I). These results indicate that GSTO2 expression was silenced by DNA hypermethylation in LSCC.

| Forced GSTO2 expression inhibited cell growth in vitro and in vivo
To clarify the function of GSTO2, we prepared GSTO2-transfected When GSTO2 transfectants were injected intravenously into nude mice, the number of hepatic tumors was lower than that in mocktransfected cells ( Figure 2E). Collectively, these results demonstrate that GSTO2 expression inhibits LSCC growth in vitro and in vivo.

| Forced GSTO2 expression inhibited the expression of β-catenin via p38 phosphorylation
Our previous study demonstrated that GSTO2 regulates β-catenin expression via p38 phosphorylation in esophageal squamous cell carcinoma (ESCC). 21 Because β-catenin has diverse physiological functions, including involvement in many cancer-related processes, 26 we next examined whether GSTO2 expression affects p38 phosphorylation and β-catenin expression in LSCC. In both cell lines used, p38 phosphorylation was accelerated in GSTO2-transfected cells compared to that in mock-transfected cells ( Figure 3A). Furthermore, we examined the association between GSTO2 expression and p38 phosphorylation in normal bronchial epithelium and detected phosphorylated p38 in airway basal cells ( Figure 3B) and Clara cells ( Figure 3C), which specifically expressed GSTO2 in normal tissues ( Figure 1A-C). Moreover, overexpression of GSTO2 suppressed the protein expression of βcatenin ( Figure 3D). As expected, no changes in β-catenin mRNA expression were induced by GSTO2 overexpression (Figure 3E), which  is concordant with our previous report that GSTO2 overexpression caused post-transcriptional downregulation of β-catenin in ESCC, probably via enhanced p38 phosphorylation. 21 Therefore, we further examined the involvement of the p38 signaling pathway in the GSTO2-mediated downregulation of β-catenin in LSCC using a p38 inhibitor. When GSTO2-transfected cells were treated for 24 hours with SB203580, a specific inhibitor of the p38 MAPK pathway, β-catenin expression was restored ( Figure 3A), suggesting that the downregulation of β-catenin caused by GSTO2 overexpression is mediated by the enhancement of p38 phosphorylation. In addition, we observed a significant reduction in the nuclear expression of β-catenin upon GSTO2 overexpression in LSCC ( Figure 3D). Because nuclear β-catenin is known to act as a transcriptional factor that modulates gene expression in carcinogenesis through the induction of cancer stem cell features, bulk tumor proliferation, and EMT, 26 we speculated that GSTO2 expression inhibits LSCC growth by modulating β-catenin-dependent gene transcription. We examined whether GSTO2 expression in LSCC affects the transcription of AXIN2, VEGFα, and MMP2, the expression of which is known to be regulated by β-catenin 27 ; however, changes in the mRNA levels of these β-catenin-targeted genes in these cell lines were inconsistent. For example, in GSTO2-transfected H520 cells, AXIN2 and VEGFα mRNA expression was significantly lower than that in mock-transfected H520 cells, whereas the transcription of AXIN2 in GSTO2-transfected LK-2 was rather accelerated compared to that in mock-transfected LK-2 cells ( Figure 3F). These results collectively suggest that growth suppression caused GSTO2 expression might not result from the modulation of β-catenin-dependent gene transcription.

| Forced GSTO2 expression inhibited the mitochondrial function via p38 phosphorylation
Recently, a novel function of β-catenin in mitochondrial metabolism as a regulator of the TCA cycle and oxidative phosphorylation (OXPHOS) was reported in hepatocytes. 28 Therefore, we examined the effect of GSTO2 expression on mitochondrial metabolism in LSCC. Metabolomic analyses using the XF96 extracellular flux analyzer revealed that GSTO2 did not affect aerobic glycolysis, as assessed by ECAR in LK-2 and H520 cells. In contrast, GSTO2 overexpression in both cell lines used suppressed mitochondrial respiration (OCR; Figure 4A). Mitochondrial function was further assessed using mitochondrial membrane potential via JC-1 dye staining. In both cell lines used, GSTO2 overexpression facilitated depolarization of the mitochondria membrane ( Figure 4B). Finally, we examined the involvement of the p38 signaling pathway in the GSTO2-mediated modulation of mitochondrial function in LSCC and confirmed that SB203580 treatment of GSTO2-transfected cells restored mitochondria membrane potential; conversely, SB203580 did not affect mitochondria membrane potential in mock-transfected cells ( Figure 4B).
These results collectively suggest that GSTO2 overexpression attenuates mitochondrial membrane potential, probably via the p38/βcatenin axis.

| D ISCUSS I ON
In this study, we demonstrated for the first time that GSTO2 is expressed in the bronchial basal cells, Clara cells, and type II alveolar cells, which correspond to stem cells in the lungs, and the loss of GSTO2 in LSCC was confirmed. We also illustrated the function of GSTO2 in regulating cell growth, β-catenin expression, and mitochondrial respiration through p38 phosphorylation.
The lungs comprise a complex organ consisting of anatomically distinct regions, such as the trachea, bronchioles, and alveoli, and they contain numerous putative stem cell populations that contribute to region-specific tissue homeostasis and repair. 7 (F) Transcript levels of AXIN2, VEGF, and MMP2 were determined by quantitative RT-PCR in mock-transfected and GSTO2transfected cells. Data are presented as the mean ±SD of assays performed in triplicate. *P < .05, **P < .01, ***P < .001 but that its expression is silenced by DNA hypermethylation in LSCC.
In addition, these findings shed light on the pivotal role of GSTO2 as a regulator for β-catenin via the p38 signaling pathway. In normal lungs, GSTO2 activates p38 MAPK, resulting in β-catenin downregulation, probably via ubiquitination. By contrast, silencing of GSTO2 is caused by DNA hypermethylation in LSCC, which allows β-catenin to escape from degradation, activate mitochondrial OXPHOS, and promote energy production ( Figure 5). Our findings may have important implications for the prevention of LSCC.

ACK N OWLED G M ENTS
We thank Drs Miwa Tamura  The red line denotes mock-transfected cells, and the blue line denotes GSTO2transfected cells. The maximum OCRs are presented as the mean ±SD (right). *P < .05, **P < .01. (B) Mitochondrial membrane potential of mock-transfected and GSTO2-transfected cells after treatment with 50 µmol/L SB203580 or DMSO for 24 h was assessed using JC-1 assay. JC-1 signals per nucleus were quantified and presented as the mean ±SD of assays performed in triplicate. *P < .05, **P < .01, ***P < .001 F I G U R E 5 Role of GSTO2 in normal lung and LSCC cells. In normal lung cells, GSTO2 activates the p38 mitogen-activated protein kinase (MAPK), which results in decrease β-catenin expression probably via ubiquitination. By contrast, silencing of GSTO2 is caused by DNA hypermethylation in LSCC, which allows β-catenin to escape from degradation, activate mitochondrial OXPHOS, and promote energy production