High glucose drives non-small cell lung cancer progression via OGT-mediated O-GlcNAcylation and PI3K/AKT/mTOR pathway activation

Abstract

Background

Hyperglycemia is an independent risk factor for poor prognosis in non-small cell lung cancer (NSCLC), yet the underlying metabolic-oncogenic crosstalk remains incompletely defined. The hexosamine biosynthetic pathway (HBP) senses glucose flux and generates UDP-GlcNAc for O-GlcNAcylation, a post-translational modification catalyzed by O-GlcNAc transferase (OGT). Here we investigate whether and how high glucose exploits OGT-mediated O-GlcNAcylation to fuel NSCLC malignancy.

Methods

A549 and H1299 NSCLC cells were cultured in normal (5.5 mM) or high (25 mM) glucose. Proliferation, colony formation, migration, invasion, wound healing, and apoptosis were evaluated. OGT and global O-GlcNAc levels were assessed in tumor versus adjacent normal tissues by immunohistochemistry and in cells by western blotting. Stable OGT-knockdown (sh-OGT) lines were generated to dissect functional requirements. PI3K/AKT/mTOR pathway activation was examined by phospho-specific western blotting. The cell lines were tested and confirmed to be mycoplasma-free.

Results

High glucose significantly enhanced proliferation, clonogenicity, migration, and invasion, and suppressed apoptosis by downregulating Bax and upregulating Bcl-2. OGT and global O-GlcNAcylation were markedly upregulated in NSCLC tissues and in high-glucose-treated cells. OGT silencing abrogated high-glucose-induced malignant phenotypes, restored apoptotic sensitivity, and reverted the Bax/Bcl-2 ratio. Mechanistically, high-glucose-driven O-GlcNAcylation activated PI3K, AKT, and mTOR phosphorylation, whereas OGT knockdown blunted this activation.

Conclusions

OGT-orchestrated hyper-O-GlcNAcylation is a pivotal conduit linking the hyperglycemic microenvironment to PI3K/AKT/mTOR-dependent NSCLC progression. Targeting OGT-mediated O-GlcNAcylation may offer a precision therapeutic strategy for NSCLC patients with comorbid diabetes or hyperglycemia.

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Key findings

• High glucose promotes proliferation, colony formation, migration, and invasion, and suppresses apoptosis in A549 and H1299 non-small cell lung cancer (NSCLC) cells.

• O-GlcNAc transferase (OGT) and global O-GlcNAcylation are upregulated in NSCLC tissues and in high-glucose-treated NSCLC cells.

• OGT knockdown reverses high-glucose-induced malignant phenotypes, restores apoptotic sensitivity, and normalizes the Bax/Bcl-2 ratio.

• High glucose activates PI3K/AKT/mTOR signaling in an OGT-dependent manner; OGT silencing blunts this activation.

What is known and what is new?

• Hyperglycemia is an independent risk factor for poor prognosis in NSCLC. The hexosamine biosynthetic pathway senses glucose flux and generates UDP-GlcNAc for O-GlcNAcylation, a post-translational modification catalyzed by OGT. Both O-GlcNAcylation and PI3K/AKT/mTOR signaling have been implicated in cancer progression, but whether and how OGT-mediated O-GlcNAcylation links hyperglycemia to NSCLC malignancy has remained incompletely defined.

• This study demonstrates that high glucose exploits OGT-mediated O-GlcNAcylation to drive NSCLC progression. OGT and global O-GlcNAc levels are elevated in NSCLC tissues and high-glucose-treated cells. Thus, OGT-orchestrated hyper-O-GlcNAcylation is a pivotal conduit linking the hyperglycemic microenvironment to PI3K/AKT/mTOR-dependent NSCLC progression.

What is the implication, and what should change now?

• These findings support further preclinical and clinical evaluation of OGT/O-GlcNAc as prognostic biomarkers and druggable targets, including combination strategies with PI3K/AKT/mTOR inhibitors. Hyperglycemia and diabetes status should be considered in NSCLC patient stratification and management, and future studies should validate these mechanisms in in vivo models and patient cohorts.

Introduction

Lung cancer remains the leading cause of cancer-related mortality worldwide, with non-small cell lung cancer (NSCLC) accounting for approximately 85% of all lung cancer cases (1). Despite significant advances in targeted therapies and immunomodulatory approaches over the past decade, the 5-year survival rate for NSCLC patients remains suboptimal (2), underscoring an urgent need to elucidate the molecular mechanisms that drive tumor progression and treatment resistance.

Emerging epidemiological evidence has identified diabetes mellitus (DM) as a significant risk factor for multiple malignancies, including NSCLC. Patients with diabetes exhibit a significantly higher risk of death from NSCLC compared with non-diabetic counterparts [hazard ratio (HR) =1.28, 95% confidence interval (CI): 1.14–1.44] (3). Hyperglycemia, the hallmark of DM, has been shown to promote malignant phenotypes in various cancer types through mechanisms that remain incompletely understood. The hexosamine biosynthetic pathway (HBP), a glucose-sensitive metabolic branch that diverts approximately 2–5% of cellular glucose flux, generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc)—the essential substrate for O-GlcNAcylation (4). This post-translational modification, catalyzed by O-GlcNAc transferase (OGT) and reversed by O-GlcNAcase (OGA), involves the addition of a single N-acetylglucosamine moiety to serine and threonine residues of nuclear and cytoplasmic proteins, functioning as a critical nutrient sensor that links metabolic status to cellular signaling networks (4,5).

Accumulating evidence has implicated aberrant O-GlcNAcylation in cancer pathogenesis. OGT and global O-GlcNAcylation levels are frequently upregulated in multiple malignancies, including NSCLC, where they have been associated with aggressive tumor behavior and poor prognosis (6). Mechanistically, OGT has been shown to promote NSCLC progression through diverse signaling axes. Recent studies have demonstrated that OGT arginine methylation by CARM1 stabilizes OGT and enhances glycolysis in NSCLC cells (7), while O-GlcNAcylation of NRF2 at Ser103 promotes lung cancer malignancy and cisplatin resistance (8). Additionally, OGT-mediated O-GlcNAcylation of transketolase (TKT) supports glycolytic flux and cellular proliferation in NSCLC (9), and OGT has been shown to activate the ERK/JUNB signaling pathway to drive NSCLC proliferation (10). OGT-regulated O-GlcNAcylation also promotes migration and invasion through activation of IL-6/STAT3 signaling (11), and the epigenetic co-regulator HCF-1 has been identified as a critical O-GlcNAcylation-dependent driver of lung cancer progression (12).

Despite these advances, the relationship between the hyperglycemic microenvironment, O-GlcNAcylation signaling, and NSCLC progression remains insufficiently explored. Elucidation of this mechanistic axis may inform novel therapeutic strategies for NSCLC patients with comorbid diabetes or hyperglycemia. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-2076/rc).

Methods

Cell lines and culture conditions

The human NSCLC cell lines A549 and H1299 were procured from the American Type Culture Collection (ATCC, Manassas, VA, USA). A549 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), whereas H1299 cells were maintained in RPMI-1640 medium, both supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). All cells were incubated at 37 ℃ in a humidified atmosphere with 5% CO₂. To simulate euglycemic and hyperglycemic microenvironments, cells were exposed to media containing either 5.5 mM (normal glucose, NG) or 25 mM (high glucose, HG) D-glucose for all subsequent functional assays.The cell lines were tested and confirmed to be mycoplasma-free.

Cell proliferation and viability assay

Cell viability was evaluated using the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Kumamoto, Japan). Cells were seeded into 96-well plates at 2×10³ cells per well and allowed to attach overnight. Following culture in normal glucose (NG) or high glucose (HG) media, 10 μL of CCK-8 reagent was added to each well at daily intervals and incubated for 2 hours at 37 ℃. Absorbance was recorded at 450 nm using a microplate reader (BioTek, Winooski, VT, USA). All experiments were performed in triplicate and repeated independently three times. Data are expressed as mean optical density ± standard deviation (SD).

Colony formation assay

For clonogenic assessment, cells were seeded in six-well plates at 500 cells per well and cultured under NG or HG conditions for 14 days, with medium replacement every three days. Colonies were fixed with 4% paraformaldehyde for 15 minutes at ambient temperature, stained with 0.5% crystal violet solution for 20 minutes, and washed thoroughly with phosphate-buffered saline (PBS). Colonies containing ≥50 cells were enumerated under a light microscope. Results are presented as mean colony counts ± SD from three independent experiments.

Migration and invasion assays

Cell migration and invasion were assessed using Transwell chambers equipped with 8-μm pore polycarbonate membranes (Corning Inc., Corning, NY, USA). For migration assays, 5×10⁴ cells suspended in 200 μL serum-free NG or HG medium were seeded into the upper compartment, with 600 μL complete medium containing 10% FBS placed in the lower chamber as a chemoattractant. For invasion assays, Transwell membranes were pre-coated with 50 μL Matrigel (1 mg/mL; BD Biosciences, Franklin Lakes, NJ, USA) prior to cell seeding. Following 24 hours of incubation at 37 ℃, non-migrated or non-invaded cells on the upper membrane surface were gently removed with cotton swabs. Cells that had traversed to the lower surface were fixed, stained with 0.1% crystal violet, and photographed in five randomly selected fields at 100× magnification under an inverted microscope. Quantitative data represent mean cell counts ± SD from three independent experiments.

Wound healing assay

Directional cell migration was further evaluated using a scratch wound healing assay. Cells were cultured in six-well plates to full confluence, and a uniform linear wound was generated by scraping the monolayer with a sterile 200-μL pipette tip. Cellular debris was removed by PBS washing, and cells were subsequently incubated in serum-free NG or HG medium. Wound images were captured at 0 and 24 hours using a phase-contrast microscope (Olympus, Tokyo, Japan) at 100× magnification. Wound closure was quantified using ImageJ software (NIH, Bethesda, MD, USA), and the relative migration rate was calculated as: [(wound area at 0 h − wound area at 24 h) / wound area at 0 h] × 100%.

Protein extraction and Western blotting

Whole-cell lysates were prepared using ice-cold RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with protease and phosphatase inhibitor cocktails. Protein concentrations were determined using the BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). Equal amounts of protein (30–40 μg per lane) were resolved by SDS-PAGE (8–12% gels) and electrotransferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA). Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 hour at room temperature and probed overnight at 4°C with primary antibodies against OGT (1:1,000; Cell Signaling Technology, Danvers, MA, USA, 24083), pan-O-GlcNAc (RL2, 1:1,000; Abcam, Cambridge, UK, ab2739), O-GlcNAc (CTD110.6, 1:1,000; Cell Signaling Technology, Danvers, MA, USA), Bax (1:1,000; Cell Signaling Technology, Danvers, MA, USA, 2772), Bcl-2 (1:1,000; Cell Signaling Technology, Danvers, MA, USA, 4223), p-PI3K (Tyr458/199, 1:1,000; Cell Signaling Technology, Danvers, MA, USA, 4228), PI3K (1:1,000; Cell Signaling Technology, Danvers, MA, USA, 4249), p-AKT (Ser473, 1:1,000; Cell Signaling Technology, Danvers, MA, USA, 4060), AKT (1:1,000; Cell Signaling Technology, Danvers, MA, USA, 9272), p-mTOR (Ser2448, 1:1,000; Cell Signaling Technology, Danvers, MA, USA, 5536), mTOR (1:1,000; Cell Signaling Technology, Danvers, MA, USA, 2983), and β-actin (1:5,000; Abcam, Cambridge, UK, ab8226). After washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL) substrate and quantified by densitometry using ImageJ software (NIH, Bethesda, MD, USA), with β-actin serving as the internal loading control.

Flow cytometric apoptosis analysis

Apoptosis was quantified using the Annexin V-FITC/propidium iodide (PI) Apoptosis Detection Kit (BD Biosciences, Franklin Lakes, NJ, USA) per the manufacturer’s protocol. Cells cultured under NG or HG conditions were harvested, washed twice with ice-cold PBS, and resuspended in 1× binding buffer at 1×10⁶ cells/mL. Cell suspensions were incubated with 5 μL Annexin V-FITC and 5 μL PI in the dark for 15 minutes at room temperature. Samples were immediately analyzed on a FACSCanto II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), and data were processed using FlowJo software (version 10.8; Tree Star, Ashland, OR, USA). The apoptotic fraction was defined as the sum of early (Annexin V⁺/PI⁻) and late (Annexin V⁺/PI⁺) apoptotic populations.

Clinical tissue specimens

Formalin-fixed, paraffin-embedded (FFPE) tissue sections containing matched tumor (T) and adjacent non-tumorous (N) lung tissues were obtained from NSCLC patients who underwent curative surgical resection. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of The Affiliated Hospital of Qingdao University (No. QYFYWZLL50490) and all specimens were collected with written informed consent.

Immunohistochemistry

IHC was performed on 4-μm FFPE sections. Following deparaffinization and rehydration, heat-induced antigen retrieval was carried out in citrate buffer (pH 6.0) at 95 ℃ for 20 minutes. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide in methanol for 15 minutes, and non-specific binding was blocked with 5% normal goat serum for 30 minutes. Sections were incubated overnight at 4 ℃ with anti-O-GlcNAc (CTD110.6, 1:200; Cell Signaling Technology, Danvers, MA, USA) and anti-OGT (1:200; Abcam, Cambridge, UK) primary antibodies. After PBS washes, sections were incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. Immunoreactivity was visualized using 3,3'-diaminobenzidine (DAB) chromogen, and sections were counterstained with hematoxylin. Images were captured at 100× and 200× magnification using a light microscope (Olympus, Tokyo, Japan). Staining was evaluated independently by two blinded pathologists.

Lentiviral-mediated OGT knockdown

Stable OGT-knockdown A549 and H1299 cell lines were generated via lentiviral transduction. Short hairpin RNA (shRNA) sequences targeting human OGT (sh-OGT) and a non-targeting scrambled control (sh-NC) were cloned into the pLKO.1 lentiviral vector (Addgene, Watertown, MA, USA). Lentiviral particles were produced by co-transfecting HEK293T packaging cells with shRNA constructs and packaging plasmids psPAX2 and pMD2.G using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA). Viral supernatants were collected at 48 and 72 hours post-transfection, filtered through 0.45-μm membranes, and used to transduce target cells in the presence of 8 μg/mL polybrene (Sigma-Aldrich, St. Louis, MO, USA). Stably transduced cells were selected with 2 μg/mL puromycin (Sigma-Aldrich, St. Louis, MO, USA) for 14 days. Knockdown efficiency was confirmed by Western blotting prior to functional experiments.

Functional assays in OGT-knockdown cells

For CCK-8 proliferation assays, sh-NC and sh-OGT cells were seeded into 96-well plates at 2×10³ cells per well and cultured under NG (sh-NC), HG (sh-NC), or HG (sh-OGT) conditions. Cell viability was measured daily for four consecutive days. Colony formation, Transwell migration, Matrigel invasion, wound healing, and flow cytometric apoptosis assays were performed as described above using sh-NC and sh-OGT cells cultured under HG conditions. Western blotting for apoptotic markers (Bax, Bcl-2) and PI3K/AKT/mTOR pathway components was conducted on lysates from sh-NC and sh-OGT cells under NG and HG conditions as described earlier.

Statistical analysis

All experiments were performed with a minimum of three independent biological replicates. Quantitative data are expressed as mean ± SD. Comparisons between two groups were analyzed using Student’s unpaired two-tailed t-test. For multiple group comparisons, one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was applied. All statistical analyses were conducted using GraphPad Prism 9.0 (GraphPad Software, USA). A P value <0.05 was considered statistically significant (P<0.05; P<0.01; P<0.001).

Results

High glucose promotes proliferation, migration, and invasion of NSCLC cells

To determine the impact of hyperglycemia on malignant behaviors, A549 and H1299 cells were cultured under normal (5.5 mM) or high (25 mM) glucose conditions. CCK-8 assays revealed that HG significantly enhanced cell viability in both lines, with statistically significant differences in A549 (P<0.01) and H1299 (P<0.001) (Figure 1A). Consistent with these findings, further CCK-8 analysis using a gradient of glucose concentrations demonstrated a dose-dependent increase in the proliferative capacity of both A549 and H1299 cells as glucose levels elevated from 5.5 to 25 mM, with growth promotion observed at intermediate clinically relevant concentrations (Figure S1). This growth advantage was further substantiated by colony formation assays, wherein HG exposure led to a marked increase in the number of crystal violet-stained colonies after 14 days (P<0.01) (Figure 1B). In addition, Transwell migration and Matrigel invasion assays demonstrated that HG potently stimulated cell motility and invasiveness, as evidenced by a substantially higher number of traversed cells compared with NG controls (P<0.01) (Figure 1C). The wound healing assay corroborated these findings, showing that HG-treated monolayers achieved significantly accelerated scratch closure at 24 hours relative to NG-treated counterparts in both A549 (P<0.01) and H1299 (P<0.001) cells (Figure 1D). Collectively, these functional assays establish that elevated glucose concentration robustly augments proliferative, clonogenic, migratory, and invasive capacities in NSCLC cells.

Figure 1.

High glucose promotes proliferation, migration, and metastasis while suppressing apoptosis in NSCLC cells. (A) Cell viability of A549 and H1299 cells cultured in NG (5.5 mM) or HG (25 mM) medium was assessed by CCK-8 assay. (B) Representative images of crystal violet-stained colonies (left) and quantification of colony numbers (right) formed by A549 and H1299 cells (200×). (C) Representative photomicrographs (left) and quantification (right) of Transwell migration (upper panels) and Matrigel invasion (lower panels) assays performed in A549 and H1299 cells under NG or HG conditions. Cells were allowed to migrate or invade for 24 hours; scale bars, 200 μm. The purple-blue spots shown in (C) represent crystal violet staining (commonly used at a concentration of 0.1–0.5%). After the migration or invasion assay, the membrane-penetrating cells were fixed with paraformaldehyde and then stained with crystal violet solution. After washing away the unbound dye, the cells appeared dark purple under the microscope and were subsequently photographed and counted. (D) Representative wound healing images of A549 and H1299 monolayers at 0 and 24 hours post-scratch under NG or HG conditions (left), and quantification of relative wound closure rates at 24 hours (right). (E) Representative Western blot images showing the expression of pro-apoptotic protein Bax, anti-apoptotic protein Bcl-2 in A549 and H1299 cells cultured under NG or HG conditions. (F) Representative flow cytometric dot plots of Annexin V-FITC/PI-stained A549 and H1299 cells cultured under NG or HG conditions (left), and quantification of total apoptotic rates (right). Data are presented as mean ± SD from three independent experiments. All P values were determined by Student’s two-tailed unpaired t-test. **, P<0.01; ***, P<0.001. CCK-8, Cell Counting Kit-8; HG, high glucose; NG, normal glucose; NSCLC, non-small cell lung cancer; OD, optical density; PI, propidium iodide; SD, standard deviation.

High glucose attenuates apoptosis by shifting the Bax/Bcl-2 balance

To elucidate the survival-promoting mechanisms underlying hyperglycemia-induced aggressiveness, the expression of key apoptosis-regulating proteins was examined. Western blot analysis revealed that HG treatment downregulated the pro-apoptotic protein Bax while concurrently upregulating the anti-apoptotic protein Bcl-2 in both A549 and H1299 cells, resulting in a marked reduction in the Bax/Bcl-2 ratio (Figure 1E). This molecular shift toward an anti-apoptotic state was functionally validated by Annexin V-FITC/PI flow cytometry, which demonstrated that HG significantly decreased the overall apoptotic fraction relative to NG controls in A549 (P<0.05) and H1299 (P<0.001) (Figure 1F). These data indicate that high glucose effectively suppresses programmed cell death through modulation of Bcl-2 family proteins, thereby contributing to enhanced cell survival and the aggravated malignant phenotype observed under hyperglycemic conditions.

O-GlcNAcylation and OGT expression are upregulated in NSCLC tumor tissues

To evaluate the clinical relevance of O-GlcNAc signaling in lung cancer, immunohistochemical staining for O-GlcNAc and O-GlcNAc transferase (OGT) was performed on paired tumor (T) and adjacent non-tumorous (N) tissue sections from NSCLC patients. As shown in (Figure 2A), O-GlcNAc immunoreactivity was predominantly detected in the cytoplasm and nuclei of tumor cells, with minimal staining in normal lung parenchyma; tumor specimens consistently exhibited strong, diffuse brown chromogenic signals, whereas adjacent tissues displayed weak or absent staining. Parallel IHC analysis revealed that OGT protein expression was also markedly elevated in tumor tissues relative to matched normal controls (Figure 2B), with robust cytoplasmic and nuclear positivity observed across multiple tumor specimens. These concordant findings demonstrate that both global O-GlcNAcylation and its catalytic enzyme OGT are upregulated in NSCLC tumor tissues, implicating enhanced hexosamine biosynthetic pathway (HBP) flux as a characteristic metabolic alteration in the NSCLC microenvironment.

Figure 2.

O-GlcNAcylation and OGT are upregulated in NSCLC tissues and are positively regulated by glucose concentration in NSCLC cell lines. (A) Representative IHC images of O-GlcNAc staining in paired adjacent non-tumorous and tumor tissue sections from NSCLC patients. Brown chromogenic signal indicates positive immunoreactivity; sections were counterstained with hematoxylin. Two representative paired specimens are shown for each group. Scale bars are indicated in the photomicrographs. (B) Representative IHC images of OGT protein expression in matched adjacent normal and tumor tissue sections from NSCLC patients. Strong cytoplasmic and nuclear OGT immunopositivity is observed in tumor tissues compared to the weak or absent staining in adjacent non-tumorous counterparts. Two representative paired specimens are shown. Scale bars are indicated. (C) Representative Western blot analysis of OGT (110 kDa) and global O-GlcNAcylation (pan-O-GlcNAc) levels in A549 and H1299 cells cultured under NG (5.5 mM) or HG (25 mM) conditions for 48 hours. Data shown are representative of three independent experiments. HG, high glucose; IHC, immunohistochemical; N, adjacent non-tumorous tissue; NG, normal glucose; NSCLC, non-small cell lung cancer; OGT, O-GlcNAc transferase; T, tumor tissue.

High glucose directly induces O-GlcNAcylation and OGT upregulation in NSCLC cell lines

To determine whether hyperglycemia per se drives O-GlcNAc signaling, A549 and H1299 cells were cultured under normal (5.5 mM) or high (25 mM) glucose conditions for 48 hours, and whole-cell lysates were analyzed by Western blotting. As depicted in Figure 2C, OGT protein levels (110 kDa) were visibly elevated in HG-treated cells compared with NG controls in both cell lines (Figure 2C). Concurrently, global O-GlcNAcylation was enhanced under HG conditions. These results establish that elevated extracellular glucose is sufficient to upregulate both OGT expression and overall O-GlcNAc modification levels in NSCLC cells, providing a direct mechanistic link between hyperglycemia and the augmentation of O-GlcNAc signaling observed in the tumor microenvironment.

OGT silencing abrogates high glucose-enhanced proliferation, clonogenicity, migration, and invasion

To determine whether OGT-mediated O-GlcNAcylation is functionally required for the pro-tumorigenic effects of hyperglycemia, stable OGT-knockdown (sh-OGT) and scramble control (sh-NC) A549 and H1299 cell lines were established. CCK-8 assays revealed that the pronounced proliferative advantage conferred by high glucose (25 mM + sh-NC) was substantially diminished upon OGT depletion (25 mM + sh-OGT), with significant differences in both cell lines (P<0.001) (Figure 3A). Colony formation assays further demonstrated that OGT knockdown markedly reduced the number and size of colonies formed under high-glucose conditions compared with sh-NC controls in A549 (P<0.01) and H1299 cells (P<0.001) (Figure 3B), effectively abrogating the clonogenic survival benefit induced by hyperglycemia. In addition, Transwell migration and Matrigel invasion assays showed that sh-OGT cells exhibited dramatically fewer migrated and invaded cells relative to sh-NC cells under HG conditions (P<0.001 for migration, P<0.01 for invasion) (Figure 3C). Wound healing assays corroborated these findings, with sh-OGT monolayers displaying significantly impaired scratch closure at 24 hours compared with sh-NC counterparts (P<0.001) (Figure 3D,3E). Collectively, these data establish that OGT is an indispensable mediator of the enhanced proliferative, clonogenic, migratory, and invasive phenotypes driven by high-glucose exposure in NSCLC cells.

Figure 3.

OGT knockdown reverses high glucose-induced proliferation, migration, invasion, and apoptosis resistance, and suppresses activation of the PI3K/AKT/mTOR signalling pathway in NSCLC cells. (A) CCK-8 proliferation curves of A549 (left) and H1299 (right) stable cell lines under three experimental conditions: normal glucose with scramble control (5.5 mM + sh-NC), high glucose with scramble control (25 mM + sh-NC), and high glucose with OGT knockdown (25 mM + sh-OGT). (B) Representative images of crystal violet-stained colonies (left) and quantification of relative migration rates (right) from colony formation assays performed in A549 and H1299 sh-NC and sh-OGT cell lines under the indicated glucose conditions for 14 days. (C) Representative photomicrographs and quantification of Transwell migration (upper panels) and Matrigel invasion (lower panels) assays in sh-NC and sh-OGT A549 and H1299 cells cultured under HG conditions. Scale bars, 200 μm. (D,E) Representative wound healing images and quantification of sh-NC and sh-OGT A549 and H1299 monolayers photographed at 0 and 24 hours post-scratch under HG conditions. Red dashed lines demarcate wound edges at each time point. (F,G) Representative Annexin V-FITC/PI flow cytometric dot plots and quantification of H1299 (upper) and A549 (lower) sh-NC and sh-OGT cells cultured under HG conditions. (H) Representative Western blot analysis showing protein expression levels of OGT (110 kDa), global O-GlcNAcylation, pro-apoptotic Bax (21 kDa), anti-apoptotic Bcl-2 (26 kDa), and loading control β-actin (42 kDa) in A549 and H1299 cells under the three indicated experimental conditions (5.5 mM + sh-NC, 25 mM + sh-NC, and 25 mM + sh-OGT). (I) Representative Western blot analysis of PI3K/AKT/mTOR pathway activation in A549 and H1299 cells under the indicated conditions. Protein levels of phosphorylated and total forms of mTOR (289 kDa), PI3K (85 kDa), and AKT (60 kDa), as well as OGT and β-actin (42 kDa), are shown. Molecular weights are indicated on the right (kDa). All data represent mean ± SD from n=3 independent experiments. P values were determined by two-way ANOVA (A), Student’s two-tailed unpaired t-test (E,G), or one-way ANOVA with Tukey’s post hoc test (B,C) as appropriate. *, P<0.05; **, P<0.01; ***, P<0.001. ANOVA, analysis of variance; CCK-8, Cell Counting Kit-8; HG, high glucose; NC, negative control; NSCLC, non-small cell lung cancer; OD, optical density; OGT, O-GlcNAc transferase; PI, propidium iodide; SD, standard deviation.

OGT depletion restores apoptotic sensitivity and modulates bcl-2 family protein expression

Flow cytometric analysis using Annexin V-FITC/PI staining was performed to evaluate whether OGT knockdown reverses the anti-apoptotic effect of high glucose. As shown in Figure 3F,3G, the overall apoptotic fraction was significantly elevated in sh-OGT cells compared with sh-NC cells under HG conditions in both A549 (P<0.01) and H1299 (P<0.01) lines, indicating that OGT-mediated hyper-O-GlcNAcylation actively suppresses programmed cell death. Western blot analysis confirmed that HG treatment increased OGT expression and global O-GlcNAcylation, accompanied by a decrease in pro-apoptotic Bax and an increase in anti-apoptotic Bcl-2 relative to NG controls (Figure 3H). Notably, OGT silencing substantially reduced OGT levels and global O-GlcNAc modification, while concurrently reversing the Bax/Bcl-2 ratio—i.e., upregulating Bax and downregulating Bcl-2—under hyperglycemic conditions. These molecular alterations provide a mechanistic explanation for the enhanced apoptosis observed in sh-OGT cells and highlight the critical role of OGT-regulated O-GlcNAcylation in shaping the apoptotic balance within the hyperglycemic tumor microenvironment.

OGT-driven hyper-O-GlcNAcylation activates the PI3K/AKT/mTOR signaling axis

Given the known crosstalk between O-GlcNAcylation and oncogenic signaling cascades, we investigated whether the PI3K/AKT/mTOR pathway is involved in mediating the pro-tumorigenic effects of OGT under high glucose. Western blot analysis (Figure 3I) revealed that high glucose treatment significantly increased the phosphorylation levels of the core upstream and downstream cascade molecules p-PI3K, p-AKT, and p-mTOR in both A549 and H1299 cells compared to normal glucose controls. Meanwhile the total protein expression of these kinases remained unchanged. Notably OGT knockdown substantially attenuated this high glucose induced phosphorylation across all three pathway components. These findings demonstrate that OGT-mediated hyper-O-GlcNAcylation functions as an upstream activator of the PI3K/AKT/mTOR cascade in the context of hyperglycemia, and that the malignant phenotypes conferred by high glucose are, at least in part, executed through this signaling axis. Collectively, our results establish OGT as a central node linking glucose metabolism to oncogenic signaling and tumor progression in NSCLC.

Discussion

In the present study, we provide comprehensive evidence establishing that a high-glucose microenvironment promotes malignant progression in NSCLC through OGT-mediated O-GlcNAcylation and subsequent activation of the PI3K/AKT/mTOR signaling pathway. Our findings demonstrate that (I) high glucose significantly enhances NSCLC cell proliferation, clonogenic survival, migration, and invasion while suppressing apoptosis; (II) OGT expression and global O-GlcNAcylation are markedly upregulated in NSCLC tumor tissues and in cells exposed to high glucose; (III) genetic knockdown of OGT abrogates high glucose-induced malignant phenotypes and restores apoptotic sensitivity; and (IV) OGT-driven hyper-O-GlcNAcylation activates the PI3K/AKT/mTOR signaling axis, identifying OGT as a critical molecular node linking glucose metabolism to oncogenic signaling in NSCLC.

Our observation that high glucose promotes NSCLC cell proliferation, migration, and invasion aligns with findings from recent studies in other malignancy types. In oral squamous cell carcinoma, Zhu et al. demonstrated that a high-glucose microenvironment accelerates malignant progression through O-GlcNAcylation, with DM status significantly associated with advanced clinical T stage, lymph node metastasis, and poor prognosis (13). Similarly, hyperglycemia has been shown to promote breast cancer proliferation and metastasis (14) and pancreatic cancer cell progression (15). These convergent findings across tumor types suggest that hyperglycemia-driven O-GlcNAcylation represents a conserved mechanism of metabolic reprogramming that confers aggressive phenotypes. The clinical relevance of our findings is underscored by meta-analysis data demonstrating that diabetic NSCLC patients exhibit a significantly higher risk of mortality (3), reinforcing the notion that glycemic status may serve as an important prognostic determinant. However, our static in vitro culture conditions rely on constant glucose concentrations, which cannot fully replicate the dynamic glycemic variability and blood glucose fluctuation characteristics typically observed in clinical diabetic patients. Future studies employing intermittent high glucose models are warranted to better simulate these complex in vivo metabolic dynamics.

A central finding of our study is the marked upregulation of OGT and global O-GlcNAcylation in NSCLC tumor tissues relative to adjacent normal tissues, as well as in high glucose-treated NSCLC cells. These observations are consistent with a growing body of literature documenting elevated O-GlcNAc modification in lung cancer (6). Importantly, we extend these findings by establishing a direct causal link between extracellular glucose concentration and OGT expression—a relationship that has been mechanistically elucidated by Lin et al., who demonstrated that CARM1 senses changes in extracellular glucose levels and mediates OGT arginine methylation at residue 348, promoting OGT stability through USP9X binding (7). This regulatory axis may explain how hyperglycemia sustains elevated OGT levels and O-GlcNAcylation in the tumor microenvironment. Additionally, HCF-1, an OGT substrate and epigenetic co-regulator, has been shown to be significantly upregulated in lung cancer tissues coincident with increased OGT and O-GlcNAc levels, and HCF-1 depletion reduces O-GlcNAcylation and proliferation in NSCLC cells (12). These observations position OGT as a central hub integrating glucose availability, protein modification, and transcriptional regulation.

The functional consequences of OGT knockdown in our study—namely, the attenuation of high glucose-induced proliferation, migration, invasion, and the restoration of apoptosis—corroborate and extend previous reports on the oncogenic role of OGT in NSCLC. OGT has been shown to promote migration and invasion through activation of IL-6/STAT3 signaling (11), and O-GlcNAcylation of PGK1 at T255 has been identified as a critical modification supporting NSCLC cell proliferation, colony formation, migration, and invasion (16). Moreover, OGT knockdown has been demonstrated to inhibit NSCLC cell proliferation and clonogenicity in vitro and tumor growth in vivo through suppression of the ERK/JUNB signaling axis (10). Our findings add to this mechanistic landscape by demonstrating that OGT is specifically required for the pro-tumorigenic effects of the hyperglycemic microenvironment, suggesting that strategies targeting OGT may be particularly beneficial for NSCLC patients with diabetes or poorly controlled glycemia.

The anti-apoptotic effect of high glucose, mediated through downregulation of Bax and upregulation of Bcl-2, was effectively reversed by OGT knockdown in our study. This is consistent with the established role of O-GlcNAcylation in modulating apoptotic pathways (17). O-GlcNAcylation has been shown to intricately modify cellular processes including apoptosis, and targeting O-GlcNAcylation has emerged as a potential strategy to overcome cancer therapeutic resistance (17). The shift in Bax/Bcl-2 balance toward an anti-apoptotic state under hyperglycemic conditions, and its reversal upon OGT depletion, provides a molecular explanation for the survival advantage conferred by the high-glucose microenvironment and highlights OGT as a potential target for sensitizing NSCLC cells to apoptosis under metabolic stress.

Perhaps most significantly, we demonstrate that OGT-driven hyper-O-GlcNAcylation activates the PI3K/AKT/mTOR signaling pathway under high-glucose conditions. This finding is supported by emerging evidence that O-GlcNAcylation crosstalks with the PI3K/AKT/mTOR cascade—one of the most frequently dysregulated oncogenic pathways in human cancers. O-GlcNAcylation of progranulin at Thr272 has been shown to activate the PI3K/AKT/mTOR pathway and drive hepatocellular carcinoma proliferation (18), and O-GlcNAcylation of DDX46 promotes hepatocellular carcinoma progression through PI3K/Akt signaling activation (19). In the context of NSCLC, OGT modifies glycolytic enzymes, transcription factors, and components of the PI3K/AKT/mTOR pathway to promote glycolysis (9,16). Our data establish that hyper-O-GlcNAcylation functions as an upstream activator of this cascade in the hyperglycemic microenvironment, with OGT knockdown substantially attenuating HG-induced phosphorylation of PI3K, AKT, and mTOR. This mechanistic insight is consistent with the findings of Zhu et al. in OSCC, where hyper-O-GlcNAcylation-mediated pro-tumoral properties partially depended on the PI3K/AKT/mTOR pathway (13), and with studies demonstrating that high glucose induces PI3Kβ binding to OGT in glioblastoma cells (20). However, the specific O-GlcNAc modification sites on these key pathway proteins remain unidentified. Determining whether this is a direct molecular mechanism requires comprehensive liquid chromatography tandem mass spectrometry and extensive site directed mutagenesis validation. Given the preliminary exploratory nature of our current study, these detailed molecular interactions have not been deeply investigated. Collectively, these data suggest that the PI3K/AKT/mTOR pathway serves as a critical downstream effector through which OGT-driven O-GlcNAcylation transduces metabolic signals into oncogenic outputs.

The therapeutic implications of our findings are substantial. OGT and O-GlcNAc signaling have been positioned as compelling targets in cancer research, and targeting OGT may suppress cancer progression across multiple malignancy types. In lung adenocarcinoma, elevated O-GlcNAcylated TR11B correlates with advanced disease, and genetic deletion of Ogt suppresses tumor development in mouse models (21). Moreover, FDA-approved drugs such as celecoxib have been shown to inhibit O-GlcNAcylation and exert antitumor effects (22). For NSCLC patients with comorbid diabetes, maintaining glycemic control may represent a clinically accessible strategy to mitigate OGT-driven tumor progression. Metformin, a widely used antidiabetic agent, has been shown to inhibit O-GlcNAcylation and OGT expression, thereby suppressing cancer cell proliferation and promoting apoptosis (23). However, it is important to note that O-GlcNAcylation is also essential for normal cellular physiology, necessitating the development of approaches that selectively target O-GlcNAcylation on specific substrates rather than globally disrupting this modification. Emerging strategies including RNA aptamers, nanobody-OGT/OGA tools, and competitive peptides offer promising avenues for site-specific manipulation of O-GlcNAcylation (24).

Several limitations of our study should be acknowledged. First, our in vitro experiments were conducted using two NSCLC cell lines (A549 and H1299). Although these models provided critical insights into cellular behaviors and basic molecular signaling, they cannot fully capture the complex systemic metabolic interactions of the tumor microenvironment. We have not yet established a mouse model of diabetes combined with NSCLC to verify these mechanistic findings in vivo. Consequently the therapeutic efficacy of targeting OGT under diabetic conditions remains to be fully elucidated in a living organism. Additionally, systematic studies are required to unravel the precise complete cascade mechanisms such as the potential competitive interplay between O-GlcNAcylation and phosphorylation at identical or adjacent amino acid residues on PI3K/AKT/mTOR pathway. Finally, although we demonstrated the upregulation of OGT and global O-GlcNAcylation in NSCLC tissues on paired tumor and adjacent non-tumor sections, our clinical analysis is currently restricted by the lack of comprehensive patient metadata. Detailed clinical information regarding the patients’ specific blood glucose levels, diabetic comorbidities, precise clinical staging, and long-term prognostic outcomes was unavailable for this specific sample cohort. Consequently we were unable to perform deep clinical stratification or evaluate the prognostic predictive value of OGT expression at this stage. Future multi-center clinical studies utilizing large patient cohorts with complete survival and metabolic datasets are urgently needed to fully elucidate the clinical prognostic significance of OGT in NSCLC.

Conclusions

In conclusion, our study identifies OGT-mediated O-GlcNAcylation as a critical mechanistic link between the hyperglycemic microenvironment and NSCLC malignant progression. High glucose upregulates OGT expression and global O-GlcNAcylation, which in turn activate the PI3K/AKT/mTOR signaling pathway to promote proliferation, migration, and invasion while suppressing apoptosis. These findings establish OGT as a central node integrating glucose metabolism with oncogenic signaling and position O-GlcNAcylation as a promising therapeutic target for NSCLC patients, particularly those with comorbid diabetes or hyperglycemia. Future investigations directed toward elucidating substrate-specific O-GlcNAcylation events and developing selective inhibitors of this modification may yield novel therapeutic strategies to improve outcomes in this patient population.

Supplementary

The article’s supplementary files as

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. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of The Affiliated Hospital of Qingdao University (No.QYFYWZLL50490) and all specimens were collected with written informed consent.

Data Sharing Statement

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DOI: 10.21037/tcr-2026-2076