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

5MPN effectively targets PFKFB4 and inhibits the glucose metabolism process and invasion of glioblastoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article 5MPN effectively targets PFKFB4 and inhibits the glucose metabolism process and invasion of glioblastoma Chen Yu, Zhennan Tao, Tianyu Lu, Hongbin Ni, Weibang Liang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7189054/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Gliomas are the most common malignant tumors of the central nervous system, with GBM (glioblastoma multiforme) being the most malignant and having the poorest prognosis. Moreover, there are very few targeted therapeutic drugs available. Tumor glucose metabolism is distinct from that of normal tissue cells and is often referred to as aerobic glycolysis. While many studies have focused on glucose metabolism in GBM, research on PFKFB4, a key regulatory kinase, is limited. Our study indicates that PFKFB4 holds therapeutic potential for targeting GBM, and we selected 5MPN, a specific PFKFB4 inhibitor that has not been studied in GBM, as the focus of this research. Our results show that 5MPN effectively inhibits glycolysis and invasion in GBM cells, and we also explored potential molecular mechanisms that may work synergistically with PFKFB4. This study will provide more options for targeted therapeutic strategies in GBM. Glioblastoma PFKFB4 5MPN glucose metabolism invasion Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Glioma is the most common malignant tumor in the central nervous system, with glioblastoma multiforme (GBM) defined as 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 produce only a small amount of ATP, providing nutrients for tumor development[ 6 ]. PFKFB4 (phosphofructokinase-2/fructose-2,6-bisphosphatase 4) 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, nor are there targeted in vitro experimental studies. 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. Materials and Methods Data collection and analysis 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 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. The cells were cultured in high-glucose medium with 10% fetal bovine serum. CCK8 The cell suspension was evenly spread on a 96-well plate with a volume of approximately 100μL and cultured overnight to allow the cells to adhere. The culture medium in the wells was removed, and 100μL of drug-containing solution was added, with the 5MPN（MCE，HY-123981） dilution concentrations being: 0, 0.5, 1, 2, 5, 10, 20, and 50μM. The cells were cultured for 24 hours and 48 hours, respectively. Then, 10μL of CCK8 (Dojindo, Japan) reagent was added to each well. After incubating the plate in a 37°C incubator for 2 hours, the optical density value was measured at a wavelength of 450nm 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 2μM 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 500μL of complete medium with 10% 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, AB137785; 1:100 dilution) at 4°C overnight. The appropriate biotinylated secondary antibody (1:100 dilution) (ZSGBio, China) was then added and incubated at 37°C 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, China). Images were captured using an inverted microscope (Olympus, 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, China) and balanced. 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°C 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% BSA, the primary antibody was added, and the samples were placed in a wet box at 4°C 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 #3195), N-cadherin (CST #14215), and β-Tubulin (CST #2146). Several test kits ATP detection kit (Beyotime, S0026B) and ATP fluorescence probe (pCMV-Mito-AT1.03, Beyotime, D2606), as well as OCR detection kit (Agilent, MitoXpress Xtra), all of which were operated according to the strict instructions provided in the kit manuals. Statistical analysis The bar chart is represented by mean standard deviation from at least three experimental replicates. Most of the experiments were statistically analyzed using Student's t test. The data were analyzed by graphpad prism 6. Significance of p values were set at NS P > 0.05, *P < 0.05, **P < 0.01, ***P< 0.001. 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 glioblastoma multiforme (GBM) (Fig. 1 A). 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 (Fig. 1 B, C). 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 (Fig. 1 D-H). The PFKFB4 expression in the glioma tissue samples we collected also showed similar results (Fig. 2 A). These results suggest that PFKFB4 may be a potentially effective therapeutic target in GBM. 5MPN inhibits PFKFB4-mediated regulation of glycolysis in GBM To investigate the inhibitory effect of PFKFB4 in GBM, we performed KEGG and 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 (Fig. 3 A-B). We selected 5MPN, a novel inhibitor of PFKFB4, exhibited effective antitumor therapeutic potential in lung cancer, to explore its effect on GBM glycolysis[ 12 ]. We used the classical GBM cell lines U251 and T98 and assessed the impact of 5MPN on cell viability using the CCK8 assay, thereby determining the drug concentrations for further experiments at 2µM and 5µM (Fig. 2 B). We observed that after 24 hours of 5MPN treatment, ATP levels in GBM cells decreased in a concentration-dependent manner, and the ATP lifetime was also reduced in a concentration-dependent manner. Additionally, GBM cells transfected with an ATP fluorescent probe plasmid showed similar results after 5MPN treatment. Moreover, the oxygen consumption rate experiment indicated that 5MPN inhibited oxygen consumption in GBM cells. These results suggest that PFKFB4 is closely associated with GBM glycolysis, and 5MPN effectively inhibits ATP levels and oxygen consumption in GBM. 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 1µM and 2µM 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 (Fig. 4 A-D). 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. These results suggest that 5MPN weakens GBM cell invasion. 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 protein-protein interaction (PPI) network analysis. We found that LDHA, CA9, SLC2A1, and PDK1 may interact with PFKFB4 (Fig. 5 A, B). 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. Discussion This study evaluated the expression of PFKFB4, an important glucose metabolism regulatory protein, in gliomas, especially glioblastoma multiforme (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, 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 ]. 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 fructose-2,6-bisphosphate. Fructose-2,6-bisphosphate is a key metabolic regulator that activates phosphofructokinase-1 (PFK-1) to promote glycolysis[ 19 ]. By reducing the level of fructose-2,6-bisphosphate, 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. However, this study did not delve into its blood-brain barrier permeability or conduct in vivo experiments using xenograft models in nude mice. Further investigations are needed in the future. 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 (protein-protein interaction) network analysis to further identify potential proteins that may be modified by PFKFB4. Our results indicate that PDK1 may interact with PFKFB4. PDK1 (3-phosphoinositide-dependent protein kinase-1) is an important protein kinase that plays a key role in various biological processes, including cell signaling, cell proliferation, survival, and metabolism[ 20 ]. It is a serine/threonine kinase that primarily regulates the activity of other target proteins through phosphorylation within the cell. Therefore, we hypothesize that PFKFB4 and PDK1 may cooperate in phosphorylation, and previous studies have confirmed our hypothesis[ 21 ]. 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. Declarations Authors’ contributions Designing research studies: Chen Yu and Weibang Liang. Conducting experiments: Chen Yu. Analyzing data: Zhennan Tao and Tianyu Lu. Collecting samples: Hongbin Ni. Preparing the manuscript: Chen Yu. The authors read and approved the final manuscript. Acknowledgements This work was supported by National Natural Science Foundation of China (No.82203876), Natural Science Foundation of basic research program of Jiangsu Province, Youth Program (BK20220184) to Zhennan Tao. Declarations Availability of data and materials The datasets generated and analysed during the current study are available in the TCGA database (https://www.cancer.gov/ccg/research/genome-sequencing/tcga), CGGA database (https://www.cgga.org.cn/), UCSC Xena database (https://xena.ucsc.edu/). Further inquiries can be directed to the corresponding author. Consent to participate All participating patients gave their oral and written informed consent. Ethics and Guidelines The study was approved by the Ethics Committee of Nanjing Drum Tower Hospital, and complied with the ethical standards set out in the Declaration of Helsinki. Consent to Publish declarations All authors agree to the publication Disclosure of Potential Conflicts of Interest The authors declare no conflict of interest References Pouyan A, Ghorbanlo M, Eslami M, Jahanshahi M, Ziaei E, Salami A, Mokhtari K, Shahpasand K, Farahani N, Meybodi TE, et al. Glioblastoma multiforme: insights into pathogenesis, key signaling pathways, and therapeutic strategies. Mol Cancer. 2025;24(1):58. https://doi.org/10.1186/s12943-025-02267-0. Lai S, Li P, Liu X, Liu G, Xie T, Zhang X, Wang X, Huang J, Tang Y, Liu Z, et al. Efficacy and safety of anlotinib combined with the STUPP regimen in patients with newly diagnosed glioblastoma: a multicenter, single-arm, phase II trial. Cancer Biol Med. 2024;21(5):433-44. https://doi.org/10.20892/j.issn.2095-3941.2023.0373. Li YA, Zhao C, Ge JJ, Li C, Xue FJ, Qi SP, Zhao C, Kong CC, Zhang JP. Correction: Apatinib combined with temozolomide in diffuse midline glioma: a novel and effective therapy. 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11:58:04","extension":"html","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":83684,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7189054/v1/1f17b31e56ab5f40f8460599.html"},{"id":92591453,"identity":"51aa75c6-b880-489b-8f74-7f9cfb730be2","added_by":"auto","created_at":"2025-10-01 11:58:03","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1564472,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of PFKFB4 and its clinical prognosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. Expression of PFKFB4 in various tumors. \u003cstrong\u003eB-C\u003c/strong\u003e. Relationship between PFKFB4 expression and prognosis of GBM patients, based on TCGA and CGGA databases. \u003cstrong\u003eD\u003c/strong\u003e. Expression of PFKFB4 in different glioma subtypes based on CGGA data, including: oligodendroglioma (O), astrocytoma (A), recurrent oligodendroglioma (rO), recurrent astrocytoma (rA), anaplastic oligodendroglioma (AO), anaplastic astrocytoma (AA), recurrent anaplastic astrocytoma (rAA), GBM, and recurrent GBM (rGBM). \u003cstrong\u003eE\u003c/strong\u003e. Expression of PFKFB4 in LGG and GBM with different IDH mutation status and 1p/19q deletion status. \u003cstrong\u003eF\u003c/strong\u003e. Expression of PFKFB4 in gliomas of different WHO grades grouped by IDH mutation status. \u003cstrong\u003eG\u003c/strong\u003e. Expression of PFKFB4 in IDH mutant and wild-type gliomas. \u003cstrong\u003eH\u003c/strong\u003e. Expression of PFKFB4 in gliomas of different WHO grades based on TCGA data.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7189054/v1/a607f323b8aee3ec2acac4d3.jpg"},{"id":92591763,"identity":"829c1219-3359-4f57-8f00-f84a3171f066","added_by":"auto","created_at":"2025-10-01 12:06:03","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1995070,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of PFKFB4 in glioma tissue samples and the effect of its inhibitor 5MPN on GBM cell viability.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. Expression of PFKFB4 in clinical tissue samples of different WHO grades. \u003cstrong\u003eB\u003c/strong\u003e. Effect of 5MPN on the viability of U251 and T98 cell lines at 24-hour and 48-hour time points under different concentration gradients, detected by CCK8 assay.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7189054/v1/d6c60427051cb47d5168e575.jpg"},{"id":92591769,"identity":"7ff5e8ea-1130-411b-a07f-d74547cd14d7","added_by":"auto","created_at":"2025-10-01 12:06:03","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1016475,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKEGG and GO analysis of PFKFB4-related genes and the effect of 5MPN on glycolysis in GBM.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA,B\u003c/strong\u003e. KEGG and GO analysis bubble plots of the top 50 genes most highly correlated with PFKFB4. \u003cstrong\u003eC\u003c/strong\u003e. ATP production in different groups. \u003cstrong\u003eD\u003c/strong\u003e. Oxygen consumption rate in different groups, with the vertical axis showing average fluorescence intensity. \u003cstrong\u003eE\u003c/strong\u003e. ATP production detected by ATP fluorescence probe in different groups. \u003cstrong\u003eF\u003c/strong\u003e. Extracellular oxygen consumption in different groups, with the x-axis showing time and the y-axis showing oxygen consumption rate. *P \u0026lt; 0.05,**P \u0026lt; 0.01, ***P \u0026lt; 0.001。\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7189054/v1/75cf278556ec5ef48705d46c.jpg"},{"id":92591454,"identity":"2102281b-a7c6-4acf-b64d-24393339d336","added_by":"auto","created_at":"2025-10-01 11:58:03","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":681280,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of 5MPN on the invasion ability of GBM cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. Visualization of the invasion distance of U251 cells in different 5MPN concentration groups using wound healing assay. \u003cstrong\u003eB\u003c/strong\u003e. Transwell assay detecting the number of invading U251 cells in different groups. \u003cstrong\u003eC\u003c/strong\u003e. Quantification of invasion distance in figure A. \u003cstrong\u003eD\u003c/strong\u003e. Quantification of invading cell numbers in figure \u003cstrong\u003eB\u003c/strong\u003e. \u003cstrong\u003eE\u003c/strong\u003e. Western blotting to detect the expression of invasion-related proteins in different groups. \u003cstrong\u003eF\u003c/strong\u003e. Immunofluorescence detection of Ecadherin and Ncadherin expression in different groups.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7189054/v1/f060c21d53b1be27020524de.jpg"},{"id":92591767,"identity":"c60cca3d-4c5b-4dd3-b5f3-77384e2b6fc0","added_by":"auto","created_at":"2025-10-01 12:06:03","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1707386,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeatmap of PFKFB4-related genes, potential protein interactions, and gene correlations.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. Heatmap visualizing genes highly correlated with PFKFB4 based on PFKFB4 expression levels in GBM cell subtypes, including: classical, proneural, and mesenchymal types. \u003cstrong\u003eB\u003c/strong\u003e. PPI (protein-protein interaction) network analysis of potential proteins that may interact with PFKFB4. \u003cstrong\u003eC\u003c/strong\u003e. Gene expression correlation with PFKFB4 that may indicate potential interactions.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7189054/v1/7efd84ef70811f185a919d53.jpg"},{"id":95720586,"identity":"fc9dc774-be7c-408a-8662-d1717073de33","added_by":"auto","created_at":"2025-11-12 09:25:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7723238,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7189054/v1/aaa383a0-f085-43ea-816a-e91360436679.pdf"},{"id":92591460,"identity":"d59bf76f-5bc2-46bb-a6b2-6d315d6dd51a","added_by":"auto","created_at":"2025-10-01 11:58:03","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3192929,"visible":true,"origin":"","legend":"","description":"","filename":"WesternBlotoriginalimage.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7189054/v1/13296085fb6e19f08f90a48e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"5MPN effectively targets PFKFB4 and inhibits the glucose metabolism process and invasion of glioblastoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlioma is the most common malignant tumor in the central nervous system, with glioblastoma multiforme (GBM) defined as WHO grade 4, having a very poor prognosis and being prone to recurrence[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. 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[\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therefore, the discovery of new targeted therapies for GBM is urgent.\u003c/p\u003e\u003cp\u003eTumor 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[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. 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 produce only a small amount of ATP, providing nutrients for tumor development[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePFKFB4 (phosphofructokinase-2/fructose-2,6-bisphosphatase 4) is an enzyme that plays a key role in glucose metabolism and belongs to the PFKFB family[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. 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[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Moreover, PFKFB4 accelerates glycolysis and promotes lactate production, which may play an important role in the formation of the tumor\u0026rsquo;s acidic microenvironment[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. 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[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, there is currently no study involving effective inhibitors of PFKFB4, nor are there targeted in vitro experimental studies.\u003c/p\u003e\u003cp\u003eBased 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.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eData collection and analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA 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 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 \u0026amp;gt; 0.8. The PPI network was visualized using Cytoscape (version 3.8.1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe U251 and T98 glioma cell lines were obtained from the Shanghai Institutes for Biological Sciences. The cells were cultured in high-glucose medium with 10% fetal bovine serum.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCCK8\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cell suspension was evenly spread on a 96-well plate with a volume of approximately 100\u0026mu;L and cultured overnight to allow the cells to adhere. The culture medium in the wells was removed, and 100\u0026mu;L of drug-containing solution was added, with the 5MPN（MCE，HY-123981） dilution concentrations being: 0, 0.5, 1, 2, 5, 10, 20, and 50\u0026mu;M. The cells were cultured for 24 hours and 48 hours, respectively. Then, 10\u0026mu;L of CCK8 (Dojindo, Japan) reagent was added to each well. After incubating the plate in a 37\u0026deg;C incubator for 2 hours, the optical density value was measured at a wavelength of 450nm using an enzyme reader, and cell viability was evaluated by the difference in optical density values.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWound healing assay and trans-well experiment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor 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\u0026mu;M 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 \u0026times; 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\u0026mu;L of complete medium with 10% 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter dewaxing, rehydration, removal of endogenous peroxidase activity, antigen retrieval, and blocking of nonspecific proteins, sections were incubated with PFKFB4 primary antibody (Abcam, AB137785; 1:100 dilution) at 4\u0026deg;C overnight. The appropriate biotinylated secondary antibody (1:100 dilution) (ZSGBio, China) was then added and incubated at 37\u0026deg;C for 60 minutes. Subsequently, the sections were stained using ABC peroxidase and diaminobenzidine (ZSGBio, China). The slides were then counterstained with Mayer\u0026apos;s hematoxylin solution (Solarbio, China). Images were captured using an inverted microscope (Olympus, Japan). The human tissue samples used in this study have complied with the relevant national and institutional policies and ethical requirements.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot and Immunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor Western blot, after extracting the cell protein sample, the protein concentration was measured using the BCA kit (Beyotime, China) and balanced. 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\u0026deg;C 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% BSA, the primary antibody was added, and the samples were placed in a wet box at 4\u0026deg;C 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 #3195), N-cadherin (CST #14215), and \u0026beta;-Tubulin (CST #2146).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSeveral test kits\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eATP detection kit (Beyotime, S0026B) and ATP fluorescence probe (pCMV-Mito-AT1.03, Beyotime, D2606), as well as OCR detection kit (Agilent, MitoXpress Xtra), all of which were operated according to the strict instructions provided in the kit manuals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe bar chart is represented by mean standard deviation from at least three experimental replicates. Most of the experiments were statistically analyzed using Student\u0026apos;s t test.\u0026nbsp;The data were analyzed by graphpad prism 6. Significance of p values were set at \u003csup\u003eNS\u003c/sup\u003eP \u0026gt; 0.05, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P\u0026lt; 0.001.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eHigh expression of PFKFB4 in GBM reflects poor prognosis\u003c/h2\u003e\u003cp\u003eTo 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 glioblastoma multiforme (GBM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). 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 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). 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 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-H). The PFKFB4 expression in the glioma tissue samples we collected also showed similar results (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). These results suggest that PFKFB4 may be a potentially effective therapeutic target in GBM.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e5MPN inhibits PFKFB4-mediated regulation of glycolysis in GBM\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the inhibitory effect of PFKFB4 in GBM, we performed KEGG and 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 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). We selected 5MPN, a novel inhibitor of PFKFB4, exhibited effective antitumor therapeutic potential in lung cancer, to explore its effect on GBM glycolysis[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. We used the classical GBM cell lines U251 and T98 and assessed the impact of 5MPN on cell viability using the CCK8 assay, thereby determining the drug concentrations for further experiments at 2\u0026micro;M and 5\u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). We observed that after 24 hours of 5MPN treatment, ATP levels in GBM cells decreased in a concentration-dependent manner, and the ATP lifetime was also reduced in a concentration-dependent manner. Additionally, GBM cells transfected with an ATP fluorescent probe plasmid showed similar results after 5MPN treatment. Moreover, the oxygen consumption rate experiment indicated that 5MPN inhibited oxygen consumption in GBM cells. These results suggest that PFKFB4 is closely associated with GBM glycolysis, and 5MPN effectively inhibits ATP levels and oxygen consumption in GBM.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eInhibition of PFKFB4 effectively suppresses GBM invasion\u003c/h2\u003e\u003cp\u003eWe 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\u0026micro;M and 2\u0026micro;M 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 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-D). 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. These results suggest that 5MPN weakens GBM cell invasion.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003ePFKFB4 may interact with metabolism-related pathway proteins\u003c/h2\u003e\u003cp\u003eTo 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 protein-protein interaction (PPI) network analysis. We found that LDHA, CA9, SLC2A1, and PDK1 may interact with PFKFB4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). 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.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study evaluated the expression of PFKFB4, an important glucose metabolism regulatory protein, in gliomas, especially glioblastoma multiforme (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, indicating possible protein interactions, although deeper mechanisms still need to be explored.\u003c/p\u003e\u003cp\u003eThe 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[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Currently, PFKFB4 is highly expressed in various cancers, such as breast cancer, lung cancer, prostate cancer, and colorectal cancer[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Its characteristic high expression in GBM suggests its potential as a target for therapy. Previous research on glioma stem cells also supports our findings[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAt 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 fructose-2,6-bisphosphate. Fructose-2,6-bisphosphate is a key metabolic regulator that activates phosphofructokinase-1 (PFK-1) to promote glycolysis[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. By reducing the level of fructose-2,6-bisphosphate, 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. However, this study did not delve into its blood-brain barrier permeability or conduct in vivo experiments using xenograft models in nude mice. Further investigations are needed in the future.\u003c/p\u003e\u003cp\u003eConsidering 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 (protein-protein interaction) network analysis to further identify potential proteins that may be modified by PFKFB4. Our results indicate that PDK1 may interact with PFKFB4. PDK1 (3-phosphoinositide-dependent protein kinase-1) is an important protein kinase that plays a key role in various biological processes, including cell signaling, cell proliferation, survival, and metabolism[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. It is a serine/threonine kinase that primarily regulates the activity of other target proteins through phosphorylation within the cell. Therefore, we hypothesize that PFKFB4 and PDK1 may cooperate in phosphorylation, and previous studies have confirmed our hypothesis[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn 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.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDesigning research studies: Chen Yu and Weibang Liang. Conducting experiments: Chen Yu. Analyzing data: Zhennan Tao and Tianyu Lu. Collecting samples: Hongbin Ni. Preparing the manuscript: Chen Yu. The authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Natural Science Foundation of China (No.82203876), Natural Science Foundation of basic research program of Jiangsu Province, Youth Program (BK20220184) to Zhennan Tao.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analysed during the current study are available in the TCGA database (https://www.cancer.gov/ccg/research/genome-sequencing/tcga), CGGA database (https://www.cgga.org.cn/), UCSC Xena database (https://xena.ucsc.edu/). Further inquiries can be directed to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll participating patients gave their oral and written informed consent.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics and Guidelines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Ethics Committee of Nanjing Drum Tower Hospital, and complied with the ethical standards set out in the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agree to the publication\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure of Potential Conflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePouyan A, Ghorbanlo M, Eslami M, Jahanshahi M, Ziaei E, Salami A, Mokhtari K, Shahpasand K, Farahani N, Meybodi TE, et al. 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Oncotarget. 2017;8(39):65143-51. https://doi.org/10.18632/oncotarget.17821.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Glioblastoma, PFKFB4, 5MPN, glucose metabolism, invasion","lastPublishedDoi":"10.21203/rs.3.rs-7189054/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7189054/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGliomas are the most common malignant tumors of the central nervous system, with GBM (glioblastoma multiforme) being the most malignant and having the poorest prognosis. Moreover, there are very few targeted therapeutic drugs available. Tumor glucose metabolism is distinct from that of normal tissue cells and is often referred to as aerobic glycolysis. While many studies have focused on glucose metabolism in GBM, research on PFKFB4, a key regulatory kinase, is limited. Our study indicates that PFKFB4 holds therapeutic potential for targeting GBM, and we selected 5MPN, a specific PFKFB4 inhibitor that has not been studied in GBM, as the focus of this research. Our results show that 5MPN effectively inhibits glycolysis and invasion in GBM cells, and we also explored potential molecular mechanisms that may work synergistically with PFKFB4. This study will provide more options for targeted therapeutic strategies in GBM.\u003c/p\u003e","manuscriptTitle":"5MPN effectively targets PFKFB4 and inhibits the glucose metabolism process and invasion of glioblastoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-01 11:57:58","doi":"10.21203/rs.3.rs-7189054/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"739f4851-5e60-4fe7-8425-bf226fcfb44d","owner":[],"postedDate":"October 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-12T09:24:57+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-01 11:57:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7189054","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7189054","identity":"rs-7189054","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}