TOP2A promotes non-small cell lung cancer progression via LDHA-mediated glycolysis and histone lactylation

Introduction

Non-small cell lung cancer (NSCLC) is the predominant form of lung cancer (LC), accounting for about 85% of all cases (1). Lung adenocarcinoma (LUAD) is the most prevalent subtype of NSCLC, making up between 40 and 50 percent of all cases (2). LC generally has no obvious symptoms in the early stage of onset. Then symptoms such as irritating dry cough, breathlessness and chest tightness may gradually appear, and coughing up blood may occur (3). When respiratory function is affected, it may cause dyspnea or respiratory failure. Smoking is one of the major causes of LC (4). In addition, genetic mutations, air pollution and previous lung diseases also increase the risk of LC (5). Despite significant progress in understanding the pathogenesis of NSCLC, it remains a major public health challenge due to its high incidence, strong invasiveness and poor prognosis.

Recent studies have shown a link between glycolysis and lactation in cancer cells. Glycolysis and lactation are interrelated processes. Increased lactate production during glycolysis can accumulate lactate-derived modifications, affecting gene expression and cellular metabolism inside cancerous cells (6). Lactation, as a new epigenetic modification, has been discovered to have a significant part in many types of tumors (7). For instance, in biopsy samples of breast cancer, colorectal cancer, etc., high concentrations of lactate indicate a higher risk of metastasis, which may lead to poor prognosis for patients (8). Lactate metabolism has also been shown to be associated with several dysregulated signaling mediators, including NF-κB, AMPK, JAK/STAT, NRF2 signaling pathways, etc. (9). Studies have shown that exogenous lactate can lead to reduced glycolytic capacity of A549 cells, changes in mitochondrial metabolic substrate selection, and decreased cell proliferation and migration. Abuduwaili et al. found that circ_0008797 inhibited NSCLC proliferation, glycolysis, and metastasis by absorbing miR-301a-3p and upregulating SOCS2 expression (10). Hua et al. also demonstrated that hypoxia-induced long non-coding RNA (lncRNA) AC020978 promoted tumor growth and glycolytic metabolism by stabilizing pyruvate kinase M2 isoform (PKM2) and enhancing HIF-1α transcriptional activity, which might be a viable target for metabolic blockers in anti-tumor treatment (11). In addition, Jiang et al. found that lactate regulates cellular metabolism in NSCLC by inducing histone lactylation, thereby regulating gene expression (12). This modification affects glycolysis and mitochondrial function, leading to cancer progression, and is linked to a worse outlook in NSCLC. These studies suggest that targeted therapy and metabolic reprogramming may be crucial to the control of metabolism and development of NSCLC.

Topoisomerase 2-alpha (TOP2A) is a key enzyme involved in DNA replication, transcription, and chromosome segregation (13). According to research, TOP2A is aberrantly expressed in malignant tumors such as breast cancer, hepatocellular carcinoma, and lung cancer, and is closely related to cell proliferation (14). TOP2A is an important anticancer drug target in a clinical study and is often used as a target for topoisomerase inhibitors such as doxorubicin and etoposide (15). Over the past few years, there has been an increasing quantity of research on the role of TOP2A in tumor metabolic reprogramming, drug resistance, and tumor microenvironment regulation. For example, Tian et al. found that low-level exposure to BDE-47 promotes prostate cancer progression through a TOP2A/lactate dehydrogenase A (LDHA)/lactation positive feedback loop, in which BDE-47 enhances glycolysis and lactate production, induces H3K18la lactylation, upregulates TOP2A, and promotes cancer growth (16). Similarly, Kanagasabai et al. demonstrated that inhibition of TOP2A in oral cancer cells reduces mitochondrial metabolism without affecting glycolysis, thereby impairing cell proliferation and cancer stem cell function (17). This suggests that TOP2A inhibition could serve as a promising therapeutic strategy by targeting metabolic pathways. In addition, Zeng et al. reported that glycolysis promotes LUAD progression by affecting immune infiltration and prognosis (18). Elevated glycolytic activity is linked to poor clinical results and resistance to immunotherapy, which involves TOP2A, emphasizing its key role in metabolic reprogramming.

A study has shown that glycolytic enzymes, such as LDHA, directly affect the level of intracellular lactate by regulating the production and clearance of lactate, thereby regulating the occurrence and outcome of lactylation modification a particular amount (19). Furthermore, high expression of LDHA in cancer cells is intimately linked to the development and incidence of malignancies, further emphasizing its important role in cellular metabolism and tumor biology (20). In this study, we attempted to analyze whether TOP2A contributes to the regulation of glycolysis and histone lactylation in NSCLC cells by bioinformatics analysis of NSCLC datasets in public databases and with subsequent experimental validation. In addition, we explored the potential molecular mechanisms of TOP2A-mediated metabolic regulation by examining its interactions with LDHA and P300. Our results might offer fresh perspectives on the molecular processes behind the progression of NSCLC and may identify new therapeutic techniques for NSCLC therapy by targeting TOP2A-mediated metabolic pathways. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-702/rc).

Methods

Obtaining and analyzing the NSCLC dataset

The Cancer Genome Atlas (TCGA)-NSCLC dataset was downloaded from the ASSISTANT for Clinical Bioinformatic website (https://www.aclbi.com/static/index.html#/), including 1,017 tumor samples and 108 normal samples. The GSE33532 and GSE74706 microarray datasets retrieved from the Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/gds/) were prepared by the R program. The GSE33532 dataset includes 80 NSCLC samples and 20 control samples. The GSE74706 dataset includes 18 NSCLC samples and 18 control samples. The mean expression value of each gene probe set was calculated, the probe identifiers (IDs) were converted into symbols for genes, and then differential analysis was performed using the Limma package in R (version 3.42.2). Fold change (FC) threshold >2 was called up-regulated differentially expressed genes (DEGs), FC <0.5 was called down-regulated DEGs, and both met the P value threshold <0.05.

Constructing protein-protein interaction (PPI) networks of overlapping DEGs and identifying the hub gene

Using the bioinformatics platform (https://bioinformatics.psb.ugent.be/webtools/Venn/), we discovered overlapping up-regulated and down-regulated DEGs in the TCGA-NSCLC, GSE33532, and GSE74706 datasets. To elucidate the potential PPIs within the overlapping DEGs, our PPI network analysis was conducted with the Search Tool for Retrieving Interacting Genes (STRING, https://string-db.org/) database. The produced PPI network, which included maximal clique centrality (MCC), BottleNeck, and maximal neighborhood component (MNC), was displayed by Cytoscape, a platform for open-source network visualization software (Version 3.7.1). The hub gene TOP2A was then identified by doing a cross-analysis of the genes in the three network modules using the bioinformatics platform once again.

Expression and correlation analysis of TOP2A and LDHA in NSCLC

The manifestation of TOP2A in tumor samples and normal samples of TCGA-NSCLC, GSE33532, and GSE74706 datasets was analyzed, and the manifestation of LDHA in samples of tumors and normal of TCGA-NSCLC dataset was analyzed. Finally, the correlation between TOP2A and LDHA expression was evaluated using the ASSISTANT for Clinical Bioinformatics platform (https://www.aclbi.com/static/index.html#/).

Cell lines and culture

Lung cancer cell lines (A549, FY-XM122; NCI-H1299, FY-22FN0709; NCI-H2087, FY-22FN0370; and SK-LU-1, FY-FH910) and normal bronchial epithelial cells (16HBE, FY-K4439Z) were acquired from Shanghai Fuyu Biotechnology Co., Ltd (Shanghai, China) and maintained in Dulbecco’s Modified Eagle Medium (DMEM) added to with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cell cultures were maintained in a humidified environment with 5% CO₂ at 37 ℃. The passage of cells occurred at 80% confluence every two to three days for subculturing.

Cell treatment

In different experiments, oxamate (0, 2.5, 5, 10, and 20 mmol/L) and 2-deoxy-D-glucose (2-DG) (0, 1, 5, and 10 mmol/L) were administered to NSCLC cells at varying doses for 0, 24, 48, and 72 hours, respectively. Furthermore, sodium lactate (Nala, a lactate derivative in the form of lactate) with a 10 mmol/L concentration was also used to treat NSCLC cells for 48 h to evaluate its effects on cellular processes. Due to the simultaneous processing of all samples, randomization was not applicable in this experiment.

Cell transfection

NSCLC cells were seeded at a density of 2×10⁵ cells per well in 24-well plates for transient transfection. Overexpression of TOP2A was achieved by transfecting cells with a plasmid encoding the TOP2A gene using Lipofectamine^TM 3000 in compliance with the manufacturer’s guidelines. To induce gene knockdown, small interfering RNA (siRNA) specific for TOP2A and LDHA was transfected into NSCLC cells. After transfection, cells were kept in a culture for an appropriate time for ensure efficient overexpression or knockdown of the target gene.

RNA extraction and cDNA synthesis

As directed by the manufacturer, total RNA was extracted from NSCLC cells using TRIzol reagent (Tiangen, Beijing, China). The concentration and purity of RNA were measured using absorbance at 260/280 nm by a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). The integrity of the RNA was verified by agarose gel electrophoresis. The PrimeScript RT reagent kit (Takara, Dalian, China) was employed to reverse transcribe 1 µg of total RNA for cDNA synthesis, as directed by the manufacturer.

Quantitative real-time polymerase chain reaction (qRT-PCR)

The usual qRT-PCR experimental procedure was followed in conducting the experiment (21). The 2^−ΔΔCt technique was employed to establish the relative gene expression levels, with β-actin as the internal reference. The primer sequences listed below were employed in the amplification process: TOP2A forward: 5'-CCGTCACCATGGAAGTGTCA-3', TOP2A reverse: 5'-TGTCTGGGCGGAGCAAAATA-3'. LDHA forward: 5'-TTGTCTCTGGCAAAGTGGAT-3', LDHA reverse: 5'-CTCCATGTTCCCCAAGGACC-3'. β-actin forward: 5'-CACCATTGGCAATGAGCGGTTC-3', β-actin reverse: 5'-AGGTCTTTGCGGATGTCCACGT-3'.

Western blot (WB) assay

Protease and phosphatase inhibitors (CoWin Biosciences, Nanjing, China) were added to radio-immunoprecipitation assay (RIPA) lysis buffer (Solarbio, Beijing, China) to create protein lysates from NSCLC cells. The BCA Protein Assay Kit measured the protein concentrations (Beyotime, China). Protein (30 µg) in equal quantities was extracted by 10% SDS-PAGE and then put onto polyvinylidene difluoride (PVDF) membranes (Beyotime, Beijing, China).After blocking the membranes with 5% skim milk, primary antibodies and HRP-conjugated secondary antibodies were incubated for a whole night at 4 ℃. Primary antibodies included: TOP2A (ab52934, 1:10,000), glucose transporter 1 (GLUT1) (ab115730, 1:100,000), LDHA (ab52488, 1:5,000), hexokinase 2 (HK2) (ab209847, 1:1,000), Histone H3 (acetyl K18) (ab177870, 1:1,000), P300 (ab275378, 1:1,000) (all from Abcam, Shanghai, China); anti-L-Lactyllysine (PTM-1401RM, 1:1,000; PTM Bio Inc., Hangzhou, China); and PKM2 (15822-1-AP, 1:2,000, Wuhan Sanying, Wuhan, China). Histone H3 (ab1791, 1:1,000, Abcam, Shanghai, China) and β-actin (ab8227, 1:1,000, Abcam, Shanghai, China) were used as loading control proteins for normalization. An enhanced chemiluminescence (ECL) kit (Tiangen, Beijing, China) was employed to observe the protein bands, and a ChemiDoc imaging system (Bio-Rad, Shanghai, China) documented the images. To assess band intensity, ImageJ software (version 1.8.0) was employed.

Extracellular acidification rate (ECAR) assay

Glycolysis and oxidative phosphorylation were assessed in vitro using a Seahorse XF96 Flux Analyzer (Seahorse Biosciences, Shanghai, China), by the manufacturer’s protocol. XF96 plates were planted with 1×10⁴ cells per well. 2-DG (50 mM), oligomycin (1 µM), and glucose (10 mM) were employed in the glycolytic stress test sequentially injected to evaluate ECAR. Time (minutes) was used to record the ECAR values, which were adjusted to the total protein content in each sample and reported as proton flux (mpH/min).

Cell Counting Kit-8 (CCK-8) assay

To assess cell viability, the CCK-8 test (KeyGEN, Nanjing, China) was employed. In 96-well plates, NSCLC cells were placed at a count of 5×10³ cells per well. Each well received an addition of CCK-8 reagent after treatment and cells were cultivated for 4 h according to the manufacturer’s protocol. A microplate reader (Kehua Technology Co., Ltd., Shanghai, China) was utilized to determine absorbance at 450 nm after 0, 24, 48, and 72 hours to assess cell proliferation.

Colony formation assay

In 60-mm plates, 2,000 cells in full media were injected, and the plates were subsequently incubated for two weeks at 37 ℃ with 5% CO₂. After being fixed with methanol for 30 minutes, cells were colored with basic nitrotetrazolium blue chloride. Colonies consisting of more than 50 cells were counted, and images were captured using a chemiluminescence imager (Clinx, Shanghai, China).

Cell invasion and migration assays

In the Transwell’s upper chamber, transfected NSCLC cells were suspended in serum-free medium. Then, the medium in the Transwell’s lower chamber was supplemented with 10% FBS. Cells with moving cell membranes were incubated for a while before being fixed with 4% paraformaldehyde and stained for 20 minutes with DAPI. Finally, the number of migratory cells in the area of view was counted using inverted microscopy. The upper chamber was coated with matrigel, and the cell invasion experiments were conducted as previously reported.

Measuring glucose, lactate, and lactate dehydrogenase (LDH) toxicity in cells

In 6-well plates, the treated cells were inserted at a concentration of 1×10⁵ cells per well, and they were then incubated in full DMEM media for the whole night. Following 24 hours of treatment, the medium was collected to quantify glucose and lactate levels. Following the manufacturer’s instructions, lactate levels were ascertained using a lactate assay kit (Solarbio, Beijing, China), and glucose concentrations were assessed by a glucose detection kit (Robio Co., Ltd., Shanghai, China). Due to the manufacturer’s instructions, LDH activity was assessed using an LDH cytotoxicity test kit (Beyotime, Shanghai, China). As directed by the directions on the corresponding kit, supernatants were collected and analyzed.

Statistical analysis

R program was employed in statistical analysis. The findings are displayed by the mean ± standard deviation (SD). The researcher performing cell counting was blinded to group allocation, and each experiment was carried out in triplicate technical replicates independently. An unpaired t-test was employed for two-group comparisons, and a one-way analysis of variance (ANOVA) was performed for comparisons between multiple groups. Tukey’s test was employed for post-hoc analysis post ANOVA. P values below 0.05 were considered to be statistically significant. All experimental procedures followed the principles of the Declaration of Helsinki and its subsequent amendments.

Results

Examining of DEGs and identification of overlapping DEGs in NSCLC datasets

In the TCGA-NSCLC dataset, 1,184 upregulated and 1,950 downregulated DEGs were discovered when tumor and normal tissues were compared (Figure 1A). Similarly, 815 up-DEGs and 1,234 down-DEGs were recognized from the GSE33532 dataset (Figure 1B). Under the same setting, 2,250 downregulated DEGs and 1,551 upregulated DEGs were found in the GSE74706 dataset (Figure 1C). The overlapping DEGs between the three datasets were further analyzed using a bioinformatics platform, and 853 overlapping downregulated DEGs and 493 overlapping upregulated DEGs were found (Figure 1D,1E).

Figure 1 Screening of DEGs and identification of the hub gene in NSCLC dataset. (A-C) Identification of DEGs in the TCGA-NSCLC (A), GSE33532 (B), and GSE74706 datasets (C). The volcano plots showed the up- and down-regulated DEGs. (D,E) Venn diagrams illustrating overlapping upregulated (D) and downregulated (E) DEGs among the three datasets. The overlapping regions represent DEGs common to all datasets. (F-H) PPI network analysis using the MCC (F), MNC (G), and BottleNeck (H) algorithms. Nodes represent genes, and edges represent interactions. The top 10 DEGs were identified based on each algorithm. (I) Cross-analysis of the PPI results from the three algorithms, identifying TOP2A as the only overlapping hub gene. (J-L) Expression levels of TOP2A in tumor and normal samples across the TCGA-NSCLC (J), GSE33532 (K), and GSE74706 (L) datasets. ****, P<0.0001. DEG, differentially expressed gene; MCC, maximal clique centrality; MNC, molecular complex detection; NSCLC, non-small cell lung cancer; PPI, protein-protein interaction; Sig, significant; TCGA, The Cancer Genome Atlas; TOP2A, topoisomerase 2-alpha.

TOP2A is highly expressed in NSCLC

The top 10 genes of overlapping DEGs were subjected to PPI network analysis utilizing the MCC, MNC, and BottleNeck algorithms. Whereas the MCC method displayed 10 and 45 edges (Figure 1F), the MNC algorithm displayed 10 nodes and 40 edges (Figure 1G). 10 nodes and 25 edges were found by the BottleNeck method (Figure 1H). A crucial intersection gene, TOP2A, was found by cross-analyzing the output of the three methods (Figure 1I). According to expression analysis, tumor samples from the TCGA-NSCLC (Figure 1J), GSE33532 (Figure 1K), and GSE74706 datasets (Figure 1L) had considerably higher amounts of TOP2A. These outcomes imply that TOP2A could serve as a proto-oncogene in the pathophysiology of NSCLC.

TOP2A knockdown inhibits NSCLC cell migration, proliferation, and invasion in vitro

WB analysis showed that TOP2A expression was significantly increased in lung cancer cell lines (NCI-H1299, SK-LU-1, A549, and NCI-H2087) contrasted with normal bronchial epithelial cells (16HBE), with the highest expression levels in NCI-H1299 and A549 cells (P<0.01) (Figure 2A,2B). NCI-H1299 and A549 cells were transfected with two independent siRNAs targeting TOP2A (si-TOP2A-1 and si-TOP2A-2). The knockdown efficiency was verified by qRT-PCR (Figure 2C,2D) and WB (Figure 2E-2G). The knockdown efficiency exceeded 50% in all cases (qRT-PCR: A549: 52.59% and 67.77%; NCI-H1299: 62.89% and 78.98%; WB: A549: 57.15% and 83.70%; NCI-H1299: 60.49% and 84.30%). The results showed that both siRNAs effectively A549 and NCI-H1299 cells have lower TOP2A levels than the small interfering RNA negative control (si-NC) (P<0.05). TOP2A knockdown significantly reduced NSCLC cell viability in CCK-8 (P<0.05) (Figure 2H,2I). Consistent with the proliferation results, colony formation assays showed thatTOP2A knockdown drastically lowered A549 and NCI-H1299 cells’ capacity to form colonies (P<0.01) (Figure 2J) (P<0.01). Transwell experiments further discovered that TOP2A knockdown dramatically reduced NSCLC cells’ capacity to migrate and invade (P<0.05) (Figure 3A,3B).

Figure 2 TOP2A knockdown inhibits NSCLC cell proliferation and colony formation. (A,B) Western blot analysis showed significantly increased expression of TOP2A in lung cancer cell lines (NCI-H1299, SK-LU-1, A549, NCI-H2087) compared to normal bronchial epithelial cells (16HBE). (C,D) qRT-PCR analysis of TOP2A mRNA expression in A549 (C) and NCI-H1299 (D) cells following TOP2A knockdown (si-TOP2A-1, si-TOP2A-2). (E) Western blot analysis of TOP2A protein expression in A549 and NCI-H1299 cells after TOP2A knockdown (si-TOP2A-1, si-TOP2A-2). (F,G) Quantification of western blot data for TOP2A protein expression in A549 and NCI-H1299 cells. (H,I) CCK-8 assays showed a significant reduction in cell viability following TOP2A knockdown (si-TOP2A-1, si-TOP2A-2) in A549 and NCI-H1299 cells. (J) Colony formation assay demonstrating a marked decrease in the colony-forming ability of NCI-H1299 and A549 cells following TOP2A knockdown (si-TOP2A-1, si-TOP2A-2). *, P<0.05; **, P<0.01. CCK-8, Cell Counting Kit-8; NSCLC, non-small cell lung cancer; OD, optical density; qRT-PCR, quantitative real-time polymerase chain reaction; TOP2A, topoisomerase 2-alpha.

Figure 3 TOP2A knockdown inhibits NSCLC cell migration and invasion. (A) Representative images and quantification of migration changes in A549 and NCI-H1299 cells transfected with si-NC, si-TOP2A-1, or si-TOP2A-2 for 24 h by Transwell assay. Scale bar =50 μm. (B) Representative images and quantification of invasion changes in A549 and NCI-H1299 cells transfected with si-NC, si-TOP2A-1, or si-TOP2A-2 for 24 h by Transwell assay. Scale bar =50 μm. Cells were stained with DAPI. *, P<0.05; **, P<0.01; ***, P<0.001. NSCLC, non-small cell lung cancer; si-NC, small interfering RNA negative control; TOP2A, topoisomerase 2-alpha.

TOP2A knockdown inhibits glycolysis in NSCLC cells

Seahorse XF96 flux analysis showed that TOP2A knockdown significantly reduced ECAR levels in both cell lines, with si-TOP2A-2 showing a more pronounced effect, indicating that glycolytic activity in NSCLC cells was significantly inhibited (Figure 4A,4B). Further evaluation using glucose and lactate detection kits showed that lactate generation and glucose absorption were significantly reduced after TOP2A knockdown (P<0.05) (Figure 4C-4F). WB study of proteins involved in glycolysis, including GLUT1, LDHA, PKM2, and HK2, showed that their expression levels were significantly reduced in TOP2A-silenced NSCLC cells (P<0.05) (Figure 4G-4I). These findings imply that TOP2A could be crucial in regulating glycolytic metabolism in NSCLC. Since si-TOP2A-2 had a higher knockdown effect than si-TOP2A-1, si-TOP2A-2 was selected for further analysis in subsequent experiments.

Figure 4 TOP2A knockdown inhibits glycolysis in NSCLC cells. (A,B) ECAR changes were measured by Seahorse XF96 flux assay after A549 (A) and NCI-H1299 (B) cells were transfected with si-NC, si-TOP2A-1, or si-TOP2A-2. (C,D) Glucose uptake was measured using a glucose assay kit after A549 (C) and NCI-H1299 (D) cells were transfected with si-NC, si-TOP2A-1, or si-TOP2A-2. (E,F) Lactate production was assessed using a lactate assay kit after A549 (E) and NCI-H1299 (F) cells were transfected with si-NC, si-TOP2A-1, or si-TOP2A-2. (G) Expression levels of glycolysis-related proteins (GLUT1, LDHA, PKM2, and HK2) in NSCLC cells after transfection with si-NC, si-TOP2A-1, or si-TOP2A-2. (H,I) Bar graphs show the quantification of western blot results of GLUT1, LDHA, PKM2, and HK2 expression in NSCLC cells after TOP2A knockdown. *, P<0.05; **, P<0.01; ***, P<0.001. ECAR, extracellular acidification rate; GLUT1, glucose transporter 1; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; NSCLC, non-small cell lung cancer; PKM2, pyruvate kinase M2 isoform; si-NC, small interfering RNA negative control; TOP2A, topoisomerase 2-alpha.

Glycolytic inhibition reduces histone lactylation in NSCLC cells

To look at the impact of glycolysis on histone lactylation, WB analysis was performed to evaluate pan-lysine lactylation (Pan kla) and H3K18 lactylation (H3K18la) levels in NSCLC cells treated with the oxamate and 2-DG. Increasing concentrations of oxamate (0, 2.5, 5, 10, 20 mmol/L) resulted in a gradual reduction in H3K18la and Pan Kla levels in NSCLC cells (Figure 5A,5B). Similarly, 2-DG treatment (0, 1, 5, 10 mmol/L) resulted in a dose-dependent decrease in Pan Kla and H3K18la levels (Figure 5C,5D). LDHA is an essential enzyme for glycolysis that catalyzes the conversion of pyruvate to lactate (22). Three different siRNAs targeting LDHA, including si-LDHA-1, si-LDHA-2, and si-LDHA-3, were transfected in NCI-H1299 and A549 cells. Their effectiveness was confirmed by WB and qRT-PCR, among which si-LDHA-2 had the most significant knockdown efficiency (P<0.05) (Figure 6A-6C). As shown by WB analysis, LDHA silencing significantly reduced Pan Kla and H3K18la levels. However, these decreases were partially reversed after treatment with sodium lactate Nala (P<0.05) (Figure 6D-6F).

Figure 5 Dose-dependent effects of glycolysis inhibitors on histone lactylation in NSCLC cells. (A,B) Western blot analysis of Pan kla and H3K18la levels in A549 (A) and NCI-H1299 (B) cells treated with different concentrations of oxamate (0, 2.5, 5, 10, 20 mmol/L). Histone H3 served as a loading control. (C,D) Western blot analysis of Pan Kla and H3K18la levels in A549 and NCI-H1299 cells treated with different concentrations of 2-DG (0, 1, 5, 10 mmol/L). Histone H3 served as a loading control. 2-DG, 2-deoxy-D-glucose; H3K18la, H3K18 lactylation; NSCLC, non-small cell lung cancer; Pan kla, pan-lysine lactylation.

Figure 6 Effects of LDHA silencing combined with NaLa on histone lactylation in NSCLC cells. (A) qRT-PCR analysis of the efficiency of LDHA knockdown in NCI-H1299 and A549 cells after transfection with si-NC, si-LDHA-1, and si-LDHA-2. *, P<0.05, **, P<0.01. (B,C) Western blot analysis of LDHA protein levels in NCI-H1299 and A549 cells after transfection with si-NC, si-LDHA-1, and si-LDHA-2 and quantification results. *, P<0.05; **, P<0.01; ***, P<0.001. (D,E) Changes in LDHA protein levels in NCI-H1299 and A549 cells after LDHA knockdown (si-LDHA-2) and Nala treatment. **, P<0.01 vs. si-NC; ^#, P<0.05 vs. si-LDHA-2. (F) Changes in Pan Kla and H3K18la levels in NSCLC cells after transfection with si-NC, si-LDHA-2, and si-LDHA-2 + Nala. H3K18la, H3K18 lactylation; LDHA, lactate dehydrogenase A; NSCLC, non-small cell lung cancer; Pan kla, pan-lysine lactylation; si-NC, small interfering RNA negative control; TOP2A, topoisomerase 2-alpha.

Glycolysis inhibition suppresses NSCLC cell proliferation and colony formation

After A549 and NCI-H1299 cells were handled with oxamate (0, 5, 10, 20 mmol/L), the CCK-8 assay detected that when the oxamate content was 5 mmol/L, the cell proliferation activity did not change significantly, but the NSCLC cell activity decreased significantly with the increase of concentration (P<0.05) (Figure 7A,7B). Similarly, following administration of varying 2-DG doses, the NSCLC cell activity also decreased in a concentration gradient (P<0.05) (Figure 7C,7D). Following the transfection of NSCLC cells by si-LDHA-2, the NSCLC cell activity reduced, but this effect was attenuated by adding Nala (P<0.05) (Figure 7E,7F). Colony formation assay showed that treatment with 20 mmol/L oxamate or 5 mmol/L 2-DG significantly inhibited the colony formation of NSCLC cells (P<0.01) (Figure 7G). Similarly, the colony formation assay also detected that the number of cell colony formations decreased after silencing LDHA, while Nala partially reversed the inhibitory effect of LDHA silencing (P<0.01) (Figure 7H).

Figure 7 Glycolysis inhibition or LDHA silencing inhibits NSCLC cell proliferation and colony formation. (A,B) CCK-8 assays were used to evaluate the changes in cell proliferation of A549 and NCI-1299 cells treated with increasing concentrations of oxamate (0, 5, 10, 20 mmol/L). *, P<0.05. (C,D) CCK-8 assays were used to evaluate the changes in cell proliferation of A549 and NCI-1299 cells treated with different concentrations of 2-DG (0, 1, 5, 10 mmol/L). *, P<0.05. (E,F) CCK-8 assays were used to detect the proliferation of A549 and NCI-H1299 cells after transfection with si-LDHA-2 or combined with Nala treatment. *, P<0.05, **, P<0.01. (G) Colony formation assays demonstrated that oxamate (20 mmol/L) and 2-DG (5 mmol/L) markedly inhibited colony-forming ability in NCI-H1299 and A549 cells. Colonies were counted and represented as bar graphs. **, P<0.01; ***, P<0.001. (H) Colony assay was used to detect the colony-forming ability of lung adenocarcinoma cells (A549 and NCI-H1299) after treatment with si-LDHA-2 or combined with Nala. Quantification of colony numbers is presented as bar graphs. **, P<0.01 vs. si-NC; ^##, P<0.01 vs. si-LDHA-2. 2-DG, 2-deoxy-D-glucose; CCK-8, Cell Counting Kit-8; LDHA, lactate dehydrogenase A; Nala, N-acetyl-L-alanine; NSCLC, non-small cell lung cancer; OD, optical density; si-NC, small interfering RNA negative control; TOP2A, topoisomerase 2-alpha.

Glycolysis inhibition can suppress NSCLC cell migration and invasion

Treatment with 20 mmol/L oxamate or 5 mmol/L 2-DG dramatically decreased the migratory capacity of NSCLC cells, according to transwell tests (P<0.05) (Figure 8A). Similarly, LDHA knockdown significantly reduced cell migration (Figure 8B). Both oxamate/2-DG treatment and LDHA knockdown also markedly inhibited cell invasion (Figure 8C,8D). Nevertheless, the addition of Nala, a lactate donor, lessened these inhibitory effects (P<0.05) (Figure 8B,8D).

Figure 8 Glycolysis inhibition or LDHA silencing inhibits NSCLC cell migration and invasion. (A) Transwell assay to detect the effect of adding glycolysis inhibitors (2-DG or oxamate) on the migration of NSCLC cells. (B) Transwell assay to detect the migration changes of NSCLC cells after transfection of si-LDHA-2 alone or in combination with Nala. (C) Transwell assay to detect the effect of adding glycolysis inhibitors (2-DG or oxamate) on the invasion of NSCLC cells. (D) Transwell assay to detect the invasion changes of NSCLC cells after transfection of si-LDHA-2 alone or in combination with Nala. The left side shows representative images of migrating or invading cells, and the right side shows the relative number of migrating or invading cells after normalization. Scale bar =50 μm. Cells were stained with DAPI. *, P<0.05. 2-DG, 2-deoxy-D-glucose; LDHA, lactate dehydrogenase A; NSCLC, non-small cell lung cancer; TOP2A, topoisomerase 2-alpha.

TOP2A promotes histone lactylation in NSCLC cells

A histone acetyltransferase (HAT) called P300 catalyzes the lactylation modification of histones (23). WB examination revealed that after TOP2A knockdown, P300 protein levels were much lower in A549 and NCI-H1299 cells (P<0.05) (Figure 9A,9B). LDH detection kit detection showed that after TOP2A knockdown, the LDH activity of NSCLC cells was significantly reduced (P<0.05) (Figure 9C). In addition, the lactate detection kit showed that after TOP2A knockdown, the lactate production of NSCLC cells was also significantly reduced (P<0.05) (Figure 9D). As shown in Figure 9E,9F, after TOP2A knockdown, the H3K18la and Pan Kla levels were significantly decreased (P<0.05), while total Histone H3 remained unchanged (Figure 9G). These results imply that TOP2A knockdown regulates histone lactylation by inhibiting P300 expression and reducing lactate production in NSCLC cells.

Figure 9 TOP2A promotes histone lactylation in NSCLC cells. (A,B) Analysis of P300 protein expression in A549 and NCI-H1299 cells transfected with si-NC, si-TOP2A-1, or si-TOP2A-2. (C) Relative LDH activity in A549 and NCI-H1299 cells transfected with si-NC, si-TOP2A-1, or si-TOP2A-2. (D) Changes in lactate levels detected by lactate assay in A549 and NCI-H1299 cells transfected with si-NC, si-TOP2A-1, or si-TOP2A-2. (E,F) Western blot analysis of LDHA expression changes in A549 and NCI-H1299 cells transfected with si-NC, si-TOP2A-1, or si-TOP2A-2. (G) Western blot analysis of Pan Kla and H3K18la levels in NSCLC cells transfected with si-NC, si-TOP2A-1, or si-TOP2A-2. Histone H3 was used as a loading control. *, P<0.05. H3K18la, H3K18 lactylation; LDH, lactate dehydrogenase; NSCLC, non-small cell lung cancer; Pan kla, pan-lysine lactylation; si-NC, small interfering RNA negative control; TOP2A, topoisomerase 2-alpha.

TOP2A overexpression alleviates the effects of glycolysis inhibition in NSCLC cells

WB analysis and qRT-PCR verified that TOP2A was effectively overexpressed in A549 and NCI-H1299 cells (P<0.05) (Figure 10A-10C). Next, WB analysis showed that oxamate and 2-DG treatment reduced LDHA expression in vector control cells, but TOP2A overexpression partially reversed this reduction in both cell lines (P<0.05) (Figure 10D). Similarly, TOP2A overexpression could also partially rescue the decrease in Pan Kla and H3K18la levels caused by glycolysis inhibitors, while total histone H3 levels remained unchanged (Figure 10E). Functional experiments showed that the inclusion of oxamate and 2-DG both significantly inhibited cell proliferation (P<0.05) (Figure 11A,11B), colony formation (P<0.05) (Figure 11C,11D), migration and invasion (P<0.05) (Figure 11E,11F) in NSCLC cells. Notably, TOP2A overexpression partially attenuated the inhibitory effects of glycolysis inhibitors on these cellular processes.

Figure 10 TOP2A overexpression rescues glycolysis inhibitor-reduced histone lactylation levels in NSCLC cells. (A) qRT-PCR analysis of upregulated TOP2A mRNA expression in A549 and NCI-H1299 cells after transfection with TOP2A overexpression plasmid. (B) Western blot analysis of TOP2A protein overexpression efficiency in A549 and NCI-H1299 cells. (C) Quantification of TOP2A protein expression in A549 and NCI-H1299 cells after transfection with TOP2A overexpression plasmid. Representative Western blot images and quantification of LDHA protein levels in NSCLC cells transfected with over-LDHA and treated with glycolysis inhibitors (oxamate or 2-DG). (E) Western blot analysis of changes in Pan Kla and H3K18la levels in NSCLC cells transfected with over-LDHA treated with glycolysis inhibitors (oxamate or 2-DG). *, P<0.05. 2-DG, 2-deoxy-D-glucose; H3K18la, H3K18 lactylation; NSCLC, non-small cell lung cancer; Pan kla, pan-lysine lactylation; qRT-PCR, quantitative real-time polymerase chain reaction; TOP2A, topoisomerase 2-alpha.

Figure 11 TOP2A overexpression attenuates the antitumor effect of glycolysis inhibitors in NSCLC cells. (A,B) CCK-8 assay shows the proliferation of A549 and NCI-H1299 cells transfected with TOP2A overexpression plasmids treated with glycolysis inhibitors (oxamate or 2-DG). (C,D) Colony assay shows the proliferation of A549 and NCI-H1299 cells transfected with TOP2A overexpression plasmids treated with glycolysis inhibitors (oxamate or 2-DG). The left side shows the number of colonies, and the right side shows the bar graph quantifying the colony formation. (E,F) Transwell migration and invasion assays quantifying the migratory and invasive capabilities of A549 and NCI-H1299 cells. Scale bar =50 μm. Cells were stained with DAPI. *, P<0.05. 2-DG, 2-deoxy-D-glucose; CCK-8, Cell Counting Kit-8; NSCLC, non-small cell lung cancer; TOP2A, topoisomerase 2-alpha.

TOP2A promotes histone lactylation in NSCLC cells by regulating LDHA

We detected the manifestation of LDHA in the TCGA-NSCLC data samples, and the results shown that tumor samples have an increased copy of the gene (P<0.0001) (Figure 12A). Correlation analysis showed that TOP2A and LDHA expression were positively correlated (Figure 12B). WB and qRT-PCR verified that A549 and NCI-H1299 cells had higher levels of LDHA expression than 16HBE cells (P<0.05) (Figure 12C-12E). WB analysis further showed that LDHA knockdown dramatically decreased TOP2A protein levels. However, TOP2A overexpression partially alleviated these decreases (P<0.05) (Figure 12F-12H). Similarly, LDHA knockdown also resulted in a reduction in histone lactylation markers Pan Kla and H3K18la levels, but this decrease was partially alleviated after transfection with TOP2A overexpression (Figure 12I). Furthermore, lactate assay results showed that LDHA knockdown significantly reduced lactate levels, whereas lactate levels were partially restored after TOP2A overexpression (P<0.05) (Figure 12J). These results imply that histone lactylation and lactate generation in NSCLC cells is regulated by a regulatory axis between LDHA and TOP2A.

Figure 12 LDHA interacts with TOP2A to regulate histone lactylation and lactate production in NSCLC cells. (A) Expression levels of LDHA in tumor and normal samples from TCGA-NSCLC datasets. The x-axis represents sample groups (tumor vs. normal), and the y-axis indicates normalized LDHA expression levels. (B) Correlation analysis of TOP2A and LDHA expression in TCGA-NSCLC datasets. Correlation coefficients and P values are above the images. (C) qRT-PCR analysis of LDHA mRNA expression in 16HBE, A549, and NCI-H1299 cells. (D) Western blot analysis of LDHA protein expression in 16HBE, A549, and NCI-H1299 cells. (E) Quantification of WB results for LDHA protein expression in 16HBE, A549, and NCI-H1299 cells. (F) Western blot analysis of TOP2A and LDHA protein levels in A549 and NCI-H1299 cells transfected with si-NC, si-LDHA-2, si-LDHA-2 + Vector, or si-LDHA-2 + over-TOP2A. (G,H) Quantification of TOP2A and LDHA protein expression in A549 and NCI-H1299 cells. (I) Western blot analysis of Pan Kla and H3K18la levels in A549 and NCI-H1299 cells transfected with si-NC, si-LDHA-2, si-LDHA-2 + Vector, or si-LDHA-2 + over-TOP2A. (J) Relative lactate levels in A549 and NCI-H1299 cells transfected with si-NC, si-LDHA-2, si-LDHA-2 + Vector, or si-LDHA-2+over-TOP2A. *, P<0.05; ****, P<0.0001. LDHA, lactate dehydrogenase A; NSCLC, non-small cell lung cancer; qRT-PCR, quantitative real-time polymerase chain reaction; TCGA, The Cancer Genome Atlas; TOP2A, topoisomerase 2-alpha; WB, western blot.

Discussion

TOP2A was shown to be a hub gene in this investigation from three NSCLC datasets through the application of bioinformatics techniques. The results of the expression analysis revealed an enormous rise of TOP2A in NSCLC tumor samples. TOP2A is an enzyme essential to the processes of transcription, DNA replication, and chromosome separation, functioning to catalyze the unwinding of DNA (24). It is strongly conveyed in proliferating cells and is essential for the process of cell division. Previous studies have demonstrated the clinical significance of TOP2A in lung cancer. As demonstrated by Kou et al., TOP2A is overstated in LUAD and is linked to a poor prognosis (25). TOP2A has been demonstrated to promote the proliferation, migration, and invasion of LUAD cells by regulating the expression of CCNB1 and CCNB2. Similarly, a study by Wu et al. corroborate the finding that TOP2A is also upregulated in NSCLC and can promote cell migration, proliferation, and epithelial-mesenchymal transition (EMT) via triggering the signaling pathway of Wnt/β-catenin (26). Our results are in line with these observations. The knockdown of TOP2A in NSCLC cell lines (NCI-H1299, A549) resulted in an important reduction in cell proliferation, migration, invasion, and tumor growth in vitro, providing compelling evidence for its role in lung cancer progression.

Glycolysis is a crucial metabolic process that creates ATP, changes glucose into pyruvate, and biosynthetic intermediates (27). Important proteins participating in the process of glycolysis include LDHA, HK2, PKM2, and GLUT1 (28). These proteins are responsible for the transmembrane transport of glucose, the generation of lactate, the catalysis of the terminal reaction of glycolysis, and the initial phosphorylation of glucose, respectively (29). Glycolytic reprogramming in NSCLC promotes tumor growth, making glycolysis a promising therapeutic target. For example, Ma et al. found that HMGA1 promotes glycolysis by regulating key glycolytic proteins including GLUT1, LDHA, and PKM2 (30). The literature by Sun et al. confirmed that the traditional Chinese medicine Shenmai Injection (SMI) affects the growth and death of cancerous cells by controlling the expression amounts of glycolysis-related enzymes (such as HK2, PKM2, GLUT1, and LDHA) (31). ECAR is a key indicator of glycolytic activity, reflecting the production of lactate, a byproduct of anaerobic glycolysis (32). It is often used to assess the metabolic state of cells, especially in the context of cancer, where glycolysis is often upregulated to support rapid growth (33). This study found that TOP2A knockdown significantly inhibited glycolysis in lung adenocarcinoma cells, as manifested by reduced ECAR, reduced glucose uptake, reduced lactate production, and reduced expression of proteins linked to glycolysis, suggesting the importance of glycolysis and its related proteins in the progression and treatment of NSCLC. In line with our findings, a recent study by Kafeel et al. reported that AdipoRon, an adiponectin receptor agonist, inhibits proliferation and induces glycolytic dependence in NSCLC cells, further supporting the notion that metabolic reprogramming plays a vital role in lung cancer progression and may be a promising therapeutic target (34).

Lactylation represents a post-translational modification, whereby lactic acid groups are added to lysine residues (35). Pan Kla denotes the pervasive lactylation of lysine residues, whereas H3K18la is a specific modification targeting histone H3, influencing gene expression and facilitating cancer metabolism and progression (36). Recent studies have underscored the significance of histone lactylation in the context of diverse cancers. Chao et al. demonstrated that elevated levels of H3K18la and Pan Kla in epithelial ovarian cancer (EOC) were linked to a bad prognosis, shorter survival, tumour stage, and platinum recurrence, indicating their potential as therapeutic targets (37). Similarly, Li et al. observed that increased histone lactylation, particularly H3K18la, promoted glycolysis in pancreatic ductal adenocarcinoma (PDAC), forming a positive feedback loop that accelerated tumour progression through TTK and BUB1B activation (38). Furthermore, Zhang et al. identified elevated Pan Kla and H3K18la levels in NSCLC, which had links to poor prognosis and immune escape (39). H3K18la has been demonstrated to enhance PD-L1 expression through the POM121/MYC pathway, thereby providing a novel potential strategy for targeting lactylation and modulating immune responses in the context of NSCLC treatment.

2-DG and oxamate are two glycolysis inhibitors that are frequently employed in the investigation of lactylation modification. 2-DG competitively inhibits glucose uptake and metabolism, thereby blocking glycolysis (40). Oxamate, on the other hand, reduces lactate production by inhibiting LDHA activity (41). Sodium lactate (NaLa) is frequently employed as a lactate donor in investigations into the function of lactate in cellular metabolism and tumor progression (42). It has been demonstrated in previous studies that lactate contributes greatly to the growth of LC. Du et al. demonstrated that NaLa-induced YY1 degradation and promotion of tumor progression through the LAR motif were regulated by LDHA (43). Li et al. demonstrated that lactate plays a pivotal role in the metabolism of lung adenocarcinoma through LDHA (44). Furthermore, Gu et al. found that lactate-induced activation of cancer-associated fibroblasts and IL-8-mediated macrophage recruitment can promote the progression of lung cancer (45). This study has revealed that glycolysis regulates histone lactylation modification in LUAD cells through LDHA. Following treatment with oxamate and 2-DG, NCI-H1299 and A549 cells revealed a dose-dependent decline in Pan Kla and H3K18la levels. The knockdown of LDHA resulted in a reduction in histone lactylation, which was reversed by the supplementation of NaLa. The outcomes of the functional experiments demonstrated that the viability of NSCLC cells declined substantially when glycolysis was inhibited. Furthermore, the inhibition of glycolysis or the knockdown of LDHA was demonstrated to considerably reduce NSCLC cells’ capacity for invasion and migration, indicating that LDHA-mediated lactate production makes a significant contribution to the growth of tumors.

HAT P300 catalyzes the addition of acetyl groups to histone lysine residues, thereby inducing chromatin relaxation and gene activation (46). This modification is associated with enhanced transcriptional activity and is essential for regulating various cellular processes. It has been demonstrated that p300 can facilitate malignant behaviors through its acetyltransferase activity (47). Furthermore, p300 is implicated in tumor cell apoptosis, drug resistance, and metabolic processes. BP/p300 has been shown to activate oncogene transcription and promote cancer cell proliferation, survival, tumorigenesis, metastasis, immune evasion, and drug resistance in both cancer cells and drug-resistant cancer cells (48). The inhibition of p300 degradation has been shown to reduce the proliferation and metastasis of LC cells, thus positioning p300 as a potential tumor suppressor. Yan et al. demonstrated that hypoxia enhances glycolysis and promotes NSCLC cell migration, stemness, and invasion through SOX9 lactylation (49). The targeting of glycolysis and lactylation represents a promising therapeutic strategy for NSCLC. Similarly, our study demonstrated that TOP2A regulates histone lactylation and glycolysis in NSCLC cells. The knockdown of TOP2A was observed to result in a significant reduction in P300 levels, lactate production, and histone lactylation markers (Pan Kla and H3K18la). The overexpression of TOP2A mitigated the effects of glycolysis inhibitors and partially restored lactylation and cellular processes such as growth and migration. TOP2A and LDHA were found to interact, and LDHA knockdown reduced histone lactylation and lactate production, while TOP2A overexpression alleviated these effects. These results suggest that TOP2A regulates histone lactylation and glycolysis, affecting tumor progression in NSCLC cells.

Despite the strengths of our study, several limitations should be acknowledged. First, the colony formation assay was conducted using a single siRNA transfection for TOP2A knockdown. While this approach is commonly used in similar studies and the knockdown efficiency was validated, the transient nature of siRNA may lead to partial recovery of gene expression during the two-week incubation. Future studies using repeated transfections or stable knockdown models will be necessary to confirm the long-term effects. Second, although we demonstrated that TOP2A knockdown leads to reduced P300 protein levels, the underlying regulatory mechanism remains unclear. It is unknown whether this effect occurs through transcriptional control, modulation of protein stability, or indirect pathways. Third, given the well-established role of TOP2A in DNA replication and chromosome segregation, we cannot fully exclude the possibility that the observed effects on glycolysis and histone lactylation are secondary to reduced proliferation rather than specific regulatory functions. Additional experiments using cell cycle–matched conditions or non-proliferative models are needed to clarify this distinction. Lastly, although two independent siRNAs were used to minimize off-target effects, we did not perform rescue experiments using siRNA-resistant TOP2A constructs, which limits the ability to definitively confirm the specificity of our findings. Future work will aim to address these limitations and further elucidate the mechanistic role of TOP2A in metabolic regulation and epigenetic remodeling in NSCLC.

Conclusions

In summary, this study identified TOP2A as a significantly upregulated gene in NSCLC by comprehensive bioinformatics analysis of three datasets. TOP2A knockdown greatly decreased NSCLC cells’ ability to proliferate, migrate, and invade in vitro. Importantly, we found that ECAR levels were reduced, glucose uptake was decreased, and lactate production was reduced after TOP2A silencing, indicating that TOP2A may be involved in regulating glycolysis in NSCLC cells. In addition, we also found that TOP2A promoted histone lactylation by regulating LDHA and P300 expression. The regulatory axis between TOP2A and LDHA is essential in regulating glycolysis and histone lactylation in NSCLC cells. According to these results, focusing on the TOP2A-LDHA axis might be a viable therapeutic approach for the management of NSCLC.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-702/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-702/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-702/prf

Funding: This work is supported by the Shanghai Changning District Committee of Science and Technology of China (grant No. CNKW2022Y05).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-702/coif). The authors have no 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. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

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