5MPN effectively targets PFKFB4 and inhibits the glucose metabolism process and invasion of glioblastoma

Introduction

Glioma is the most common malignant tumor in the central nervous system, with glioblastoma multiforme (GBM) defined as World Health Organization (WHO) grade 4, having a very poor prognosis and being prone to recurrence (1). Currently, except for temozolomide, no effective chemotherapy has significantly benefited GBM patients. Adjuvant chemotherapy drugs such as anlotinib, apatinib, and bevacizumab are only effective in a small subset of GBM populations (2-4). Therefore, the discovery of new targeted therapies for GBM is urgent.

Tumor glucose metabolism is a crucial intermediate step in the biological progression of tumors, providing energy for various biological behaviors of tumors, including proliferation, invasion, migration, and metastasis (5). The glucose metabolism in tumors differs from that in normal cells, known as aerobic glycolysis, or the Warburg effect: that is, under aerobic conditions, large amounts of glucose are consumed, broken down into lactate, and only a small amount of adenosine triphosphate (ATP) are produced, providing nutrients for tumor development (6).

Phosphofructokinase-2/fructose-2,6-bisphosphatase 4 (PFKFB4) is an enzyme that plays a key role in glucose metabolism and belongs to the PFKFB family (7). It plays an important role in regulating glycolysis and gluconeogenesis. PFKFB4 activates phosphofructokinase-1 (PFK-1) by generating fructose-2,6-bisphosphate (F-2,6-BP), thereby promoting glycolysis. The enhancement of glycolysis provides more ATP and metabolic intermediates for tumor cells, which not only supply energy but also participate in the synthesis of biomolecules such as lipids and amino acids, supporting tumor cell growth and division (8). Moreover, PFKFB4 accelerates glycolysis and promotes lactate production, which may play an important role in the formation of the tumor’s acidic microenvironment (9). Recent studies have shown that in gliomas, PTBP1 glycosylation promotes the maintenance of glioma stem cells through glycolysis driven by PFKFB4; PFKFB4 also regulates the malignant progression of GBM through the AKT signaling pathway (10,11). However, there is currently no study involving effective inhibitors of PFKFB4, and there are no targeted in vitro experimental studies. We selected 5MPN, a novel inhibitor of PFKFB4, exhibited effective antitumor therapeutic potential in lung cancer, adjusted to explore its effect on GBM glycolysis (12).

Based on previous research, this study comprehensively evaluates the expression of PFKFB4 in GBM and its potential for targeted therapy. A specific inhibitor, 5MPN, was selected for in vitro experimental studies, predicting possible molecular interaction mechanisms of PFKFB4, and providing new insights for future targeted treatment strategies for GBM. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1740/rc).

Methods

Data collection and analysis

The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. RNA sequencing (RNA-seq) transcriptional data and clinical information of glioma were downloaded from The Cancer Genome Atlas (TCGA) database (https://cancergenome.nih.gov/) and the Chinese Glioma Genome Atlas (CGGA) database (https://www.cgga.org.cn/). The bar plot of differential gene expression, clinical prognosis line plot, heatmap, and correlation analysis plot were generated using R programming (version 4.4.2). The protein-protein interaction (PPI) protein network analysis was performed by incorporating these mRNAs into the PPI network using the STRING database (https://string-db.org/) with a confidence score >0.8. The PPI network was visualized using Cytoscape (version 3.8.1).

Cell culture

The U251 and T98 glioma cell lines were obtained from the Shanghai Institutes for Biological Sciences. We confirm the recent short tandem repeat (STR) analysis and mycoplasma detection of U251 and T98 cells. The cells were cultured in high-glucose medium with 10% fetal bovine serum.

Cell Counting Kit-8 (CCK8)

The cell suspension was evenly spread on a 96-well plate with a volume of approximately 100 µL and cultured overnight to allow the cells to adhere. The culture medium in the wells was removed, and 100 µL of drug-containing solution was added, with the 5MPN (MCE, NJ New Jersey, USA; HY-123981) dilution concentrations being: 0, 0.5, 1, 2, 5, 10, 20, and 50 µM. The cells were cultured for 24 hours and 48 hours, respectively. Then, 10 µL of CCK8 (Dojindo, CK18, Kyushu, Japan) reagent was added to each well. After incubating the plate in a 37 ℃ incubator for 2 hours, the optical density value was measured at a wavelength of 450 nm using an enzyme reader, and cell viability was evaluated by the difference in optical density values.

Wound healing assay and trans-well experiment

For the wound healing assay, cells were seeded in a 6-well plate and incubated overnight. The next day, the inserts were removed, and serum-free medium containing 5MPN at concentrations of 0, 1, and 2 µM was added. After 24 and 48 hours, migration distance was calculated using GraphPad. For the Transwell assay, to evaluate cell invasion ability, tumor cells (5×10⁴ cells) were seeded into the upper chamber of Transwell inserts coated with matrix gel, and serum-free medium was used. The same 5MPN concentration gradient was applied. The lower chamber contained 500 µL of complete medium with 10% fasting blood sugar (FBS) to induce cell invasion. After 24 hours of incubation, cells that migrated through the membrane were stained with 0.2% crystal violet and photographed by microscopy.

Immunohistochemistry

After dewaxing, rehydration, removal of endogenous peroxidase activity, antigen retrieval, and blocking of nonspecific proteins, sections were incubated with PFKFB4 primary antibody (Abcam, Cambridge, UK; AB137785; 1:100 dilution) at 4 ℃ overnight. The appropriate biotinylated secondary antibody (1:100 dilution) (ZSGBio, SAP-9100, Beijing, China) was then added and incubated at 37 ℃ for 60 minutes. Subsequently, the sections were stained using ABC peroxidase and diaminobenzidine (ZSGBio, China). The slides were then counterstained with Mayer’s hematoxylin solution (Solarbio, H8070, Beijing, China). Images were captured using an inverted microscope (Olympus, IX53, Tokyo, Japan). The human tissue samples used in this study have complied with the relevant national and institutional policies and ethical requirements.

Western blot and immunofluorescence

For Western blot, after extracting the cell protein sample, the protein concentration was measured using the BCA kit (Beyotime, P0010, Shanghai, China) and equalized. Then, high-temperature denaturation was performed. The samples were then added to the gel plate sample loading tank and electrophoresis was carried out at constant voltage for about 90 minutes, followed by transfer to the membrane at constant current for about 90 minutes. Next, the membrane was blocked with milk, incubated with the primary antibody at 4 ℃ overnight, and then exposed after incubation with the secondary antibody the next day. For immunofluorescence, after cell adhesion, the cells were fixed with 4% paraformaldehyde, blocked with 5% bovine serum albumin (BSA), the primary antibody was added, and the samples were placed in a wet box at 4 ℃ overnight. The next day, the samples were incubated with fluorescent secondary antibodies and sealed with DAPI mounting medium. The coverslips with attached cells were placed on adhesive slides. Finally, images were captured using a confocal microscope. The primary antibodies used included MMP2 (Abcam, AB92536), E-cadherin (CST, Boston, USA; #3195), N-cadherin (CST, #14215), and β-tubulin (CST, #2146).

Several metabolism test kits

ATP detection kit (Beyotime, S0026B) and ATP fluorescence probe (pCMV-Mito-AT1.03, Beyotime, D2606), as well as optical character recognition (OCR) detection kit (Elabscience, Wuhan, China; E-BC-F068), all of which were operated according to the strict instructions provided in the kit manuals. Among them, the detection of ATP concentration was repeated three times in the experiment. During OCR detection, the fluorescence intensity of extracellular oxygen consumption is measured every 2 minutes.

Statistical analysis

The bar chart is represented by the mean ± standard deviation from at least three experimental replicates. The number of repeated experiments involved is 3. Most of the experiments were statistically analyzed using Student’s t-test. The data were analyzed by Graphpad Prism 6. Significance of P values was set at P<0.05.

Results

High expression of PFKFB4 in GBM reflects poor prognosis

To clarify the clinical significance of PFKFB4, we analyzed its expression in different tumors using data from the TCGA database. We found that PFKFB4 is highly expressed in various tumors, with the highest expression observed in GBM (Figure 1A). In the clinical prognosis correlation analysis based on TCGA and CGGA data, we found that high expression of PFKFB4 in GBM patients reflects a poor prognosis; in CGGA data, PFKFB4 expression showed a significant negative correlation in gliomas (Figure 1B,1C). In different pathological and molecular subtypes of gliomas, we observed that PFKFB4 was most highly expressed in GBM, with more significant expression in recurrent GBM and IDH1 wild-type GBM (Figure 1D-1H). The PFKFB4 expression in the glioma tissue samples we collected also showed similar results (Figure 2A). In the detection of PFKFB4 expression positivity rate, we found that GBM (WHO grade 4) had the highest expression, and there were differences in expression between low grade (WHO grade 2), high grade (WHO grade 3), and GBM (P<0.01) (Figure 2B). These results suggest that PFKFB4 may be a potentially effective therapeutic target in GBM.

Figure 1 The expression of PFKFB4 and its clinical prognosis. (A) Expression of PFKFB4 in various tumors. (B,C) Relationship between PFKFB4 expression and prognosis of GBM patients, based on TCGA (n=168) and CGGA (n=222) databases. (D) Expression of PFKFB4 in different glioma subtypes based on CGGA data, including: O (n=48), A (n=46), rO (n=4), rA (n=10), AO (n=12), AA (n=38), rAA (n=24), GBM (n=85), and rGBM (n=24). (E) Expression of PFKFB4 in LGG and GBM with different IDH mutation status and 1p/19q deletion status. (F) Expression of PFKFB4 in gliomas of different WHO grades grouped by IDH mutation status. (G) Expression of PFKFB4 in IDH mutant and wild-type gliomas. (H) Expression of PFKFB4 in gliomas of different WHO grades based on TCGA data. Data in (D-H) are shown as Student’s t-test and Anova test, the P value of the difference is displayed above the chart. A, astrocytoma; AA, anaplastic astrocytoma; AC, adenocarcinoma; AO, anaplastic oligodendroglioma; CGGA, Chinese Glioma Genome Atlas; GBM, glioblastoma multiforme; LGG, lower-grade glioma; O, oligodendroglioma; PFKFB4, phosphofructokinase-2/fructose-2,6-bisphosphatase 4; pTPM, proteomics-informed transcripts per million; rA, recurrent astrocytoma; rAA, recurrent anaplastic astrocytoma; rGBM, recurrent GBM; rO, recurrent oligodendroglioma; SQCC, squamous cell carcinoma; TCGA, The Cancer Genome Atlas; WHO, World Health Organization.

Figure 2 Expression of PFKFB4 in glioma tissue samples and the effect of its inhibitor 5MPN on GBM cell viability. (A) Immunohistochemical staining for detecting the expression of PFKFB4 in clinical tissue samples of different WHO grades. The following image (scale bar =20 µm) is an enlarged version of the previous image (scale bar =100 µm). (B) Count the number of positive cells in a single field of view and calculate the positivity rate. Data are shown as the mean ± SD. n=3, **, P<0.01, Student’s t-test. (C) Effect of 5MPN on the viability of U251 and T98 cell lines at 24- and 48-hour time points under different concentration gradients, the dose-response curve is plotted on its right sidedetected by CCK8 assay. CCK8, Cell Counting Kit-8; GBM, glioblastoma multiforme; IC50, half-maximal inhibitory concentration; PFKFB4, phosphofructokinase-2/fructose-2,6-bisphosphatase 4; SD, standard deviation; WHO, World Health Organization.

5MPN inhibits PFKFB4-mediated regulation of glycolysis in GBM

We used the classical GBM cell lines U251 and T98 and assessed the impact of 5MPN on cell viability using the CCK8 assay. The half-maximal inhibitory concentration (IC50) values for U251 and T98 were 14.62 and 10.15 µM, respectively, and we determined drug concentrations of 2 and 5 µM for further experiments (Figure 2C). To investigate the inhibitory effect of PFKFB4 in GBM, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses on the top 50 genes most correlated with PFKFB4 based on TCGA data. We found that these genes were mainly enriched in glycolysis pathways, which is consistent with the glycolytic kinase activity of PFKFB4 itself (Figure 3A,3B). We observed that after 24 hours of 5MPN treatment, ATP levels in GBM cells decreased in a concentration-dependent manner (Figure 3C). Additionally, the oxygen consumption rate experiment indicated that 5MPN inhibited oxygen consumption in GBM cells (Figure 3D). Moreover, GBM cells transfected with an ATP fluorescent probe plasmid showed similar results after 5MPN treatment (Figure 3E). These results suggest that PFKFB4 is closely associated with GBM glycolysis, and 5MPN effectively inhibits ATP levels and oxygen consumption in GBM.

Figure 3 KEGG and GO analysis of PFKFB4-related genes and the effect of 5MPN on glycolysis in GBM. (A,B) KEGG and GO analysis bubble plots of the top 50 genes most highly correlated with PFKFB4. (C) ATP production in different groups. (D) Oxygen consumption rate in different groups, with the vertical axis showing average fluorescence intensity. (E) ATP production detected by ATP fluorescence probe in different groups. Data in (C,D) are shown as the mean ± SD. n=3. *, P<0.05; ***, P<0.001, Student’s t-test. ATP, adenosine triphosphate; GBM, glioblastoma multiforme; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; NC, negative control; PFKFB4, phosphofructokinase-2/fructose-2,6-bisphosphatase 4; SD, standard deviation.

Inhibition of PFKFB4 effectively suppresses GBM invasion

We investigated the regulatory effect of PFKFB4 on GBM invasion to evaluate the antitumor effect of 5MPN. To minimize the cytotoxicity of 5MPN on GBM cells and more accurately assess its impact on GBM cell invasion, we selected 1 and 2 µM as the concentration gradient. Our wound healing assay and trans-well assay results indicated that 5MPN inhibited the invasion distance of the cells, and the number of invasive cells significantly decreased, all of which showed a concentration-dependent manner (Figure 4A-4D). In the detection of invasion-related markers, we observed a decrease in the expression of invasion-related proteins MMP2 and N-cadherin and an increase in E-cadherin expression after 5MPN treatment, as confirmed by Western blot and immunofluorescence assays (Figure 4E,4F). These results suggest that 5MPN weakens GBM cell invasion.

Figure 4 Effect of 5MPN on the invasion ability of GBM cells. (A) Visualization of the invasion distance of U251 cells in different 5MPN concentration groups using wound healing assay. (B) Transwell assay detecting the number of invading U251 cells in different groups. Cells were stained with 0.2% crystal violet.scale bar =50 µm. (C) Quantification of invasion distance in (A). (D) Quantification of invading cell numbers in (B). (E) Western blotting to detect the expression of invasion-related proteins in different groups. (F) Immunofluorescence detection of E-cadherin and N-cadherin expression in different groups. Data in (C,D) are shown as the mean ± SD. n=3; **, P<0.01; ***, P<0.001, Student’s t-test. GBM, glioblastoma multiforme; NC, negative control; SD, standard deviation.

PFKFB4 may interact with metabolism-related pathway proteins

To further explore the regulatory mechanism of PFKFB4, we conducted an expression analysis of the top 20 genes most correlated with PFKFB4 expression. We constructed a heatmap based on PFKFB4 expression levels and the subtypes of GBM and combined it with PPI network analysis. We found that LDHA, CA9, SLC2A1, and PDK1 may interact with PFKFB4 (Figure 5A,5B). We then analyzed the correlation between the expression of PFKFB4 and these proteins, with PDK1 showing the highest correlation, suggesting that PFKFB4 may have a protein-protein interaction and regulatory relationship with PDK1 based on the assumption of bioinformatics (Figure 5C).

Figure 5 Heatmap of PFKFB4-related genes, potential protein interactions, and gene correlations. (A) Heatmap visualizing genes highly correlated with PFKFB4 based on PFKFB4 expression levels in GBM cell subtypes, including: classical, proneural, and mesenchymal types. (B) PPI network analysis of potential proteins that may interact with PFKFB4. (C) Gene expression correlation with PFKFB4 that may indicate potential interactions. GBM, glioblastoma multiforme; PFKFB4, phosphofructokinase-2/fructose-2,6-bisphosphatase 4; PPI, protein-protein interaction.

Discussion

This study evaluated the expression of PFKFB4, an important glucose metabolism regulatory protein, in gliomas, especially GBM, and analyzed its relationship with the prognosis of GBM patients. 5MPN, as the latest PFKFB4 inhibitor, was explored for its inhibitory effects on glucose metabolism and invasion in GBM, suggesting that it could be a potential drug for targeting GBM in the future. Further mechanistic investigations revealed that PFKFB4 and PDK1 expressions are most strongly correlated, based on the assumption of bioinformatics, indicating possible protein interactions, although deeper mechanisms still need to be explored.

The TCGA database contains clinical sample information and gene expression sequencing data for various cancers, while the CGGA database includes clinical data for gliomas, as well as mRNA, methylation modifications, microRNA, and other sequencing data, providing strong data support for this study (13,14). Currently, PFKFB4 is highly expressed in various cancers, such as breast cancer, lung cancer, prostate cancer, and colorectal cancer (9,15-17). Its characteristic high expression in GBM suggests its potential as a target for therapy. Previous research on glioma stem cells also supports our findings (18-20).

At present, there are no effective targeted therapies for PFKFB4 in gliomas. Among the newly developed targeted drugs, 5MPN is the only one available for PFKFB4 research. 5MPN competitively binds with the substrate of PFKFB4, inhibiting its kinase activity and reducing the synthesis of F-2,6-BP. F-2,6-BP is a key metabolic regulator that activates PFK-1 to promote glycolysis (21). By reducing the level of F-2,6-BP, 5MPN effectively inhibits the glycolytic process. This study conducted part of the preclinical research of 5MPN in GBM, marking its innovative nature. Our findings show that 5MPN effectively inhibits glycolysis and invasion in GBM cells, indicating its potent anti-tumor effects.

Considering that PFKFB4 itself is a kind of kinase with the characteristic of phosphorylating substrates as a protein modification, we generated a heatmap of genes most strongly correlated with its expression and performed a PPI network analysis to further identify potential proteins that may be modified by PFKFB4. Our results indicate that PDK1 (3-phosphoinositide-dependent protein kinase-1) may interact with PFKFB4 based on the assumption of bioinformatics.

We have pointed out some limitations of this study here. This study did not delve into the permeability of the 5MPN blood-brain barrier, nor did it conduct in vivo experiments using xenograft models in nude mice. Further investigation is needed in the future. We also overlooked the recovery experiments on the relationship between glycolysis itself and invasion, and did not validate co-immunoprecipitation (CO-IP) in the direct interaction between PFKFB4 and PDK1. We hope to improve this in further research.

In conclusion, we believe that targeting PFKFB4 in GBM is necessary for therapy, and 5MPN, by inhibiting glycolysis and invasion in GBM cells, could be a potential anti-GBM targeted drug. This study provides new insights for targeted therapy in GBM.

Conclusions

This study proposed a new drug option that may be effective for clinical treatment of GBM. We found the therapeutic potential of 5MPN in GBM and expected to conduct more in-depth research to complete clinical transformation.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1740/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1740/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1740/prf

Funding: This work was supported by National Natural Science Foundation of China (No. 82203876), and Natural Science Foundation of basic research program of Jiangsu Province, Youth Program (No. BK20220184, to Z.T.).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1740/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. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

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

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