Osteoblast-specific factor 2 reduces glutathione by downregulating PRKRA expression in oral squamous cell carcinoma

Introduction

Oral cavity carcinomas are the most frequent malignancies among head and neck cancers (1,2), of which oral squamous cell carcinoma (OSCC) accounts for 90%. OSCC is often detected at a relative advanced stage, resulting in a worse prognosis (3). As reported by the Global Cancer Observatory (GCO), the incidence of OSCC is expected to see a continual increase of 40% by 2040, alongside a rise in mortality (4). Exploring the mechanisms of OSCC would facilitate identification of prognostic factors and enhancement of therapeutic strategies for OSCC patients.

Osteoblast-specific factor 2 (OSF-2, POSTN) exhibits high expression across various cancers and drives invasion in head and neck squamous cell carcinoma (5,6). Over the past few decades, cancer has been recognized as a metabolic disorder characterized by alterations in multiple metabolic pathways, such as amino acid, fatty acid and lipid metabolism (7,8). To support the rapid growth and proliferation, cancer cells exhibit high demand for glucose and glutamine metabolism (9). Although glutamine is a nonessential amino acid (NEAA), it is considered conditionally essential in cancer cells because of their elevated demand for it (10). Glutamine plays an important role in cell metabolism, including nucleotide biosynthesis, glutathione (GSH) synthesis, and as a supplementation to tricarboxylic acid (TCA) cycle (10).

GSH, synthesized from cysteine, glutamate, and glycine, plays an important role in the protection of tumor cells against oxidative stress, such as stress induced by reactive oxygen species (ROS) (11). GSH mostly exists in the reduced form, and can be oxidized to glutathione disulfide (GSSG), the oxidized form (12). Diminished GSH levels perturb redox homeostasis, potentially inducing ROS accumulation and subsequently triggering cell death (13). Researchers have reported that GSH depletion can exacerbate oxidative stress in cancer cells and potentiate the response to cancer therapy (13). However, whether POSTN affects GSH levels in OSCC remains to be elucidated.

PACT is encoded by the PRKRA gene and belongs to the RNA-induced silencing complex (RISC). It has been reported to be dysregulated in cancers such as epithelial skin cancer and non-small cell lung carcinoma (14). PACT is a dsRNA-binding protein that binds protein kinase R (PKR) at multiple domains. It can be phosphorylated at Ser-18, Ser-246 and Ser-287, leading to PKR activation (15). Furthermore, PACT has been implicated in promoting chemoresistance in ovarian cancer as well as progression of liver cancer (16). High PACT expression also contributes to the chemoresistance of mucinous ovarian cancer (17). In breast cancer, patients with high PACT expression have a poorer prognosis than those with low PACT expression (18). However, how PACT is expressed in oral cancer is currently unknown.

In this study, we aimed to explore the metabolic status of GSH in OSCC and elucidate the mechanisms by which it is regulated. Our findings demonstrated that POSTN decreased GSH expression in oral cancer cells. Additionally, POSTN reduced PRKRA expression, whereas PRKRA overexpression enhanced GSH levels. Collectively, these findings identified a previously unrecognized mechanism governing GSH regulation in oral cancer and showed a promising therapeutic strategy for oral cancer patients. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1086/rc).

Methods

Cell culture

HN6 and Cal27 cells were obtained from the National Institutes of Health and were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA), containing 10% fetal bovine serum (FBS) (Gibco), 100 units/mL penicillin and 100 µg/mL streptomycin. All cells were cultured at 37 °C in a humidified 5% CO₂ atmosphere.

RNA sequencing

Total RNA was extracted from HN6 cells. Then the libraries were prepared in triplicate from the RNA samples according to manufacturer instructions. The RNA sequence alignment was performed, followed by data processing and significance testing.

Metabolomics analysis

The nontargeted metabolic profiling was performed using liquid chromatography-tandem mass spectrometry (LC-MS) method. The instrument was an ACQUITY ultra-performance liquid chromatography coupled with an AB SCIEX Triple TOF 5600 plus System. Mobile phase A was composed of 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. High resolution mass spectrometer can be operated and employed for the acquisition of primary and secondary mass spectrometry data.

Protein mass spectrometry

Protein samples were obtained from HN6 cells following treatment with or without recombinant human periostin (rhPOSTN). Proteins were enzymatically decomposed into polypeptides and then were used for mass spectrometry analysis. The corresponding protein database was used to analyze the mass spectrometry data and identify the proteins detected.

The detection of GSH, GSSG and ROS levels

The HN6 and Cal 27 cells were treated with rhPOSTN or lentiviral vector (LV)-PRKRA plasmid. The control group were treated without rhPOSTN or control plasmid. Intracellular concentrations of GSH and GSSG were measured using a GSSG/GSH quantification kit and ROS detection kit. All operation steps were carried out in strict accordance with the instructions. All experiments were performed with at least three biological replicates.

Western blot analysis

HN6 and Cal 27 cells were collected and lysed in RIPA lysis buffer (Yeasen, Shanghai, China), followed by protein quantification using the BCA kit (Thermo Fisher Scientific, Waltham, USA). Proteins were electrophoresed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with non-fat milk for 1 h at room temperature, and probed with primary antibodies against PRKRA (1:1,000, 10771-1-AP; Proteintech, Rosemont, USA) and β-actin (1:1,000, 66009-1-Ig; Proteintech, USA) overnight at 4 °C. Then, the membrane was incubated with corresponding rabbit (1:10,000, RGAR001; Proteintech, USA) or mouse secondary antibodies (1:10,000, RGAM001; Proteintech, USA) and visualized with ECLUltra.

Statistical analysis

The data were analyzed with SPSS 19.0 statistical software. Normality of data distribution was assessed using the Shapiro-Wilk test, and homogeneity of variances was evaluated using Levene’s test. Student’s t-test was used for comparisons between two groups, and one-way analysis of variance (ANOVA) was used for comparisons among multiple groups. P<0.05 was considered to be statistically significant.

Results

POSTN might regulate the metabolic pathways in OSCC

To explore the influence of POSTN on OSCC, transcriptome sequencing was performed on recombinant human POSTN (rhPOSTN)-stimulated OSCC cells, and the analysis identified that 650 genes were upregulated while 554 genes were downregulated in OSCC cells (Figure 1A,1B). Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, multiple metabolic pathways were found to be altered, suggesting that POSTN might be involved in metabolic regulation in OSCC (Figure 1C). Therefore, we speculated that POSTN might be a regulator of metabolic pathways in OSCC.

Figure 1 POSTN might regulate the metabolic pathways in OSCC. After HN6 cells were treated with or without rhPOSTN, the transcriptome sequencing was performed. (A,B) 650 genes were upregulated and 554 genes were downregulated; (C) multiple metabolic pathways were changed in rhPOSTN-treated HN6 cells. DEG, differentially expressed gene; FC, fold change; KEGG, Kyoto Encyclopedia of Genes and Genomes; NC, negative control; OSCC, oral squamous cell carcinoma; POSTN, osteoblast-specific factor 2; rhPOSTN, recombinant human POSTN.

POSTN might inhibit the GSH metabolism in OSCC

To investigate how POSTN regulated OSCC metabolism, metabolomics analysis was employed. The results showed that GSH metabolism pathways were markedly inhibited in OSCC cells following stimulation with rhPOSTN (Figure 2A,2B), suggesting that POSTN is a regulator of GSH metabolism. Mass spectrometric detection was subsequently performed and the characteristics of proteins and metabolic pathways were analyzed. The data revealed that GSH metabolism pathways exhibited significant alterations (Figure 2C). Together, these data indicate that POSTN might inhibit the GSH metabolism in OSCC.

Figure 2 POSTN might inhibit the glutathione metabolism in OSCC. (A,B) After HN6 cells were exposed to rhPOSTN or vehicle control, metabolomics analysis showed that the glutathione metabolism pathways were inhibited in OSCC cells. (C) The combined analysis of metabolomics analysis and mass spectrometric detection. ATP, adenosine triphosphate; GTP, guanosine triphosphate; NC, negative control; OSCC, oral squamous cell carcinoma; POSTN, osteoblast-specific factor 2.

POSTN decreased GSH expression while elevating ROS levels in OSCC cells

To further validate this finding, we examined the expression of GSH and ROS in OSCC cells. The results showed that after being treated with rhPOSTN, the expression of GSH was significantly decreased in OSCC cells. After HN6 cells were treated with 10 ng/mL rhPOSTN, the GSH expression levels significantly reduced from 0.052 to 0.042 µmol/mL (P=0.003) while the GSSG levels significantly increased from 0.5479 to 0.6583 µmol/mL (P=0.007). The expression levels of GSH in Cal 27 cells were 0.043 µmol/mL and significantly decreased to 0.037 µmol/mL following 10 ng/mL rhPOSTN treatment (P=0.004). The expression levels of GSSG in Cal 27 cells were 0.5573 µmol/mL and remarkedly increased to 0.7152 µmol/mL following 10ng/mL rhPOSTN treatment (P=0.002) (Figure 3A,3B). Besides, ROS levels were increased in both HN6 and Cal 27 cells after treatment with rhPOSTN (Figure 3C). The data revealed a non-monotonic dose-response of POSTN on GSSG levels in HN6 and Cal 27 cells. POSTN at 10 ng/mL increased both GSSG and ROS levels in HN6 and Cal 27 cells, suggesting that classical GSH oxidation occurred in OSCC cells. POSTN at 20 ng/mL elevated ROS levels, while unexpectedly reduced GSSG levels in HN6 and Cal 27 cells. This paradoxical finding suggests that higher concentrations of POSTN might promote GSSG clearance or metabolism by activating alternative pathways. Accordingly, these data suggest that POSTN decreased GSH expression while elevating ROS levels in OSCC cells.

Figure 3 POSTN decreased GSH expression while elevating ROS levels in OSCC cells. After treatment with 10 or 20 ng/mL rhPOSTN, the (A) GSH, (B) GSSG, and (C) ROS levels in HN6 and Cal 27 cells were detected. Scale bar: 1,000 µm. **, P<0.01; ***, P<0.001. GSH, glutathione; GSSG, glutathione disulfide; NC, negative control; OSCC, oral squamous cell carcinoma; POSTN, osteoblast-specific factor 2; ROS, reactive oxygen species.

POSTN suppressed GSH expression through downregulating PRKRA

The results of mass spectrometric detection showed that PRKRA was downregulated in rhPOSTN-treated cells. Western blot assay was performed to validate PRKRA expression, revealing that rhPOSTN treatment led to a significant reduction in PRKRA levels in HN6 and Cal27 cells (Figure 4A,4B). Subsequently, PRKRA was overexpressed in OSCC cells, which resulted in increased GSH levels. The data revealed that GSH levels in HN6 increased from 0.7863 µmol/mL (NC group) to 1.102 µmol/mL following PRKRA overexpression (P=0.02). Consistently, Cal27 cells in the LV-PRKRA group exhibited markedly higher GSH levels (1.114 µmol/mL) compared with those in NC group (0.7780 µmol/mL) (P=0.03). Treatment with rhPOSTN abrogated the elevation of GSH levels caused by PRKRA overexpression (Figure 4C). Conversely, GSSG levels in both HN6 and Cal 27 cells were significantly reduced when PRKRA was overexpressed (Figure 4D). Further detection revealed that POSTN increased ROS expression in HN6 and Cal 27 cells, while PRKRA overexpression led to a marked reduction in ROS levels (Figure 4E). Thus, these findings indicate that POSTN suppressed GSH expression through downregulation of PRKRA.

Figure 4 POSTN suppressed GSH expression through downregulating PRKRA. (A,B) After treatment with 10 or 20 ng/mL rhPOSTN, PRKRA expression in HN6 and Cal 27 cells were detected. After HN6 and Cal 27 cells were treated with rhPOSTN or overexpression of PRKRA, (C) GSH, (D) GSSG, and (E) ROS levels were detected. GSH, glutathione; GSSG, glutathione disulfide; LV, lentiviral vector; NC, negative control; POSTN, osteoblast-specific factor 2; rhPOSTN, recombinant human POSTN; ROS, reactive oxygen species.

The mechanism regulating GSH expression

To investigate the mechanism underlying GSH expression, the mass spectrometric detection was performed in HN6 cells. The KEGG pathway analysis identified several pathways potentially involved in POSTN induced GSH reduction, with the top three being apoptotic signaling pathways, Wnt signaling pathways and I-kappaB kinase/NF-kappaB signaling pathways (Figure 5). Additionally, phosphoproteomic analysis revealed that PRKRA was phosphorylated at S1 and S16. Therefore, we assumed that POSTN might reduce GSH expression through phosphorylation of PRKRA, which in turn downregulated PRKRA expression.

Figure 5 The KEGG pathway analysis of mass spectrometric detection in HN6 cells. KEGG, Kyoto Encyclopedia of Genes and Genomes; TOR, target of rapamycin.

Discussion

OSCC is regarded as the most common and lethal malignant neoplasm in the head and neck region (19). Although targeted therapy and immunotherapy have improved the survival rate of OSCC patients, their efficacy is still restricted to recurrent and/or metastatic cases (20). The importance of cancer cell metabolism and the limitations of conventional cancer therapies have prompted the development of novel cancer treatment strategies (21). Accumulating evidence indicates that cancer cells rely heavily on glutamine uptake, and inhibitors of glutamine metabolism can exert stronger antitumor effects when combined with immune checkpoint inhibitors (21-23). These findings suggest that GSH may play a critical role in cancer development.

In the present study, we showed that POSTN suppressed GSH metabolism and reduced PRKRA expression in OSCC cells. When PRKRA was overexpressed, the reduction in GSH levels caused by POSTN was reversed. PRKRA is involved in inflammation, a process closely linked to tumor progression (16). Additionally, PRKRA plays an oncogenic role in hepatocellular carcinoma (HCC) and is associated with poor prognosis in hepatitis B virus (HBV)-related HCC (14). Nevertheless, the expression and functional role of PRKRA in OSCC have yet to be elucidated. The present study is the first to characterize the role of PRKRA and its relationship with GSH levels.

GSH serves as a critical constituent of cellular antioxidative defense system. Generally, high levels of oxidative stress exist in cancer cells, making them more sensitive to GSH deficiency (12). GCL inhibitor buthionine sulfoximine (BSO) reduces GSH production and promotes apoptosis in breast and ovarian cancer (24). Ogiwara et al. reported that combined treatment with inhibitors targeting GSH and SLC7A11 synergistically enhanced cancer cell death (25). Studies demonstrated that GSH levels were elevated in solid tumors relative to normal tissues (26). Actually, elevated GSH levels in tumors is associated with enhanced resistance to chemotherapy and ROS-based therapies (26).

Our study showed that GSH expression was decreased in cancer cells following POSTN treatment, maybe due to enhanced GSH consumption during cancer cells proliferation and invasion. The synthesis of GSH is dependent on the availability of amino acids. When tumor cells require large numbers of amino acids for growth, GSH synthesis might be reduced, leading to lower GSH levels in tumor cells (12). Additionally, GSH can be effluxed from cells by GSH transferases (GSTs) (27). Acevedo-León et al. further demonstrated that GSH levels reduced by more than 50% in colorectal cancer, whereas GSSG levels were increased (28). In clinical studies, GSH and GSSG are commonly measured in blood, a minimally invasive method that reflects tissue redox status (29). Nevertheless, GSH and GSSG levels vary greatly among healthy patients, and the predictive value of GSH/GSSG ratio still needs further research.

Conclusions

This study showed that POSTN reduced the GSH level by downregulating the expression of PRKRA. By establishing a link between the PRKRA-GSH axis and OSCC malignancy, these findings provided insights for cancer therapeutic interventions.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1086/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1086/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1086/prf

Funding: This work was supported by Shanghai’s Top Priority Research Center (No. 2022ZZ01017) and the CAMS Innovation Fund for Medical Sciences (CIFMS) (No. 2019-I2M-5-037).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1086/coif). All authors declare that a patent application related to the work reported in this manuscript was filed in November 2023. The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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