M2 macrophages regulate KDM6B/PFKFB2 metabolic reprogramming of cervical squamous cell carcinoma through CXCL1.

Macrophages in the tumor microenvironment can polarize into M1 or M2 forms, with M2 macrophages (M2φ) promoting tumor growth and metastasis in cervical squamous cell carcinoma (CESC). This study explored the effects of M2φ on CESC metabolic reprogramming both in vitro and in vivo. Results showed that M2φ secreted CXCL1, which significantly increased CESC migration and metabolic regulation. Further experiments revealed that CXCL1 upregulated KDM6B to enhance PFKFB2 transcriptional activity, thus regulating CESC glucose metabolism. Transcriptome sequencing screened 5 upregulated genes related to glycolysis, with PFKFB2 showing the most significant increase in cells treated with rCXCL1. Dual-luciferase reporter assay confirmed that rCXCL1 enhances PFKFB2 transcriptional activity. Bioinformatics analysis revealed a high correlation between expressions of KDM6B and PFKFB2 in CESC. Mechanistic experiments demonstrated that KDM6B inhibited H3K27me3 modification to activate PFKFB2 transcriptional expression. In conclusion, M2φ secreted CXCL1 to promote CESC cell migration and invasion, and CXCL1 activated KDM6B expression in CESC cells, inhibiting H3K27 protein methylation modification, and enhanced PFKFB2 transcriptional activity to regulate CESC glucose metabolism. These results provided new insights into the complex interplay between the immune system and cancer metabolism, which may have broader implications for understanding and treating other types of cancer.

Cardiac-specific PFKFB3 overexpression prevents diabetic cardiomyopathy via enhancing OPA1 stabilization mediated by K6-linked ubiquitination.

Diabetic cardiomyopathy (DCM) is a prevalent complication of type 2 diabetes (T2D). 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) is a glycolysis regulator. However, the potential effects of PFKFB3 in the DCM remain unclear. In comparison to db/m mice, PFKFB3 levels decreased in the hearts of db/db mice. Cardiac-specific PFKFB3 overexpression inhibited myocardial oxidative stress and cardiomyocyte apoptosis, suppressed mitochondrial fragmentation, and partly restored mitochondrial function in db/db mice. Moreover, PFKFB3 overexpression stimulated glycolysis. Interestingly, based on the inhibition of glycolysis, PFKFB3 overexpression still suppressed oxidative stress and apoptosis of cardiomyocytes in vitro, which indicated that PFKFB3 overexpression could alleviate DCM independent of glycolysis. Using mass spectrometry combined with co-immunoprecipitation, we identified optic atrophy 1 (OPA1) interacting with PFKFB3. In db/db mice, the knockdown of OPA1 receded the effects of PFKFB3 overexpression in alleviating cardiac remodeling and dysfunction. Mechanistically, PFKFB3 stabilized OPA1 expression by promoting E3 ligase NEDD4L-mediated atypical K6-linked polyubiquitination and thus prevented the degradation of OPA1 by the proteasomal pathway. Our study indicates that PFKFB3/OPA1 could be potential therapeutic targets for DCM.

Targeting the reprogrammed metabolism in H3.3K27M pediatric high-grade gliomas.

Recent studies in Diffuse Midline Gliomas (DMG) demonstrated a strong connection between epigenome dysregulation and metabolic rewiring. Here, we evaluated the value of targeting a glycolytic protein named Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase 3 (PFKFB3) in H3.3K27M DMG. We observed that the viability of H3.3K27M cells is dramatically reduced by PFK15, a potent inhibitor of PFKFB3. Furthermore, PFKFB3 inhibition induced apoptosis and G2/M arrest. Interestingly, CRISPR-Knockout of the K27M mutant allele has a synergistic effect on the observed phenotype. Altogether, we identified PFKFB3 as a new target for H3.3K27M DMG, making PFK15 a potential candidate for future animal studies and clinical trials.

Context-dependent modification of PFKFB3 in hematopoietic stem cells promotes anaerobic glycolysis and ensures stress hematopoiesis.

Metabolic pathways are plastic and rapidly change in response to stress or perturbation. Current metabolic profiling techniques require lysis of many cells, complicating the tracking of metabolic changes over time after stress in rare cells such as hematopoietic stem cells (HSCs). Here, we aimed to identify the key metabolic enzymes that define differences in glycolytic metabolism between steady-state and stress conditions in murine HSCs and elucidate their regulatory mechanisms. Through quantitative 13C metabolic flux analysis of glucose metabolism using high-sensitivity glucose tracing and mathematical modeling, we found that HSCs activate the glycolytic rate-limiting enzyme phosphofructokinase (PFK) during proliferation and oxidative phosphorylation (OXPHOS) inhibition. Real-time measurement of ATP levels in single HSCs demonstrated that proliferative stress or OXPHOS inhibition led to accelerated glycolysis via increased activity of PFKFB3, the enzyme regulating an allosteric PFK activator, within seconds to meet ATP requirements. Furthermore, varying stresses differentially activated PFKFB3 via PRMT1-dependent methylation during proliferative stress and via AMPK-dependent phosphorylation during OXPHOS inhibition. Overexpression of Pfkfb3 induced HSC proliferation and promoted differentiated cell production, whereas inhibition or loss of Pfkfb3 suppressed them. This study reveals the flexible and multilayered regulation of HSC glycolytic metabolism to sustain hematopoiesis under stress and provides techniques to better understand the physiological metabolism of rare hematopoietic cells.

Multi-omics and immunogenomics analysis revealed PFKFB3 as a targetable hallmark and mediates sunitinib resistance in papillary renal cell carcinoma: in silico study with laboratory verification.

Glycolysis-related metabolic reprogramming is a central hallmark of human cancers, especially in renal cell carcinoma. However, the regulatory function of glycolytic signature in papillary RCC has not been well elucidated. In the present study, the glycolysis-immune predictive signature was constructed and validated using WGCNA, glycolysis-immune clustering analysis. PPI network of DEGs was constructed and visualized. Functional enrichments and patients' overall survival were analyzed. QRT-PCR experiments were performed to detect hub genes' expression and distribution, siRNA technology was used to silence targeted genes; cell proliferation and migration assays were applied to evaluate the biological function. Glucose concentration, lactate secretion, and ATP production were measured. Glycolysis-Immune Related Prognostic Index (GIRPI) was constructed and combined analyzed with single-cell RNA-seq. High-GIRPI signature predicted significantly poorer outcomes and relevant clinical features of pRCC patients. Moreover, GIRPI also participated in several pathways, which affected tumor immune microenvironment and provided potential therapeutic strategy. As a key glycolysis regulator, PFKFB3 could promote renal cancer cell proliferation and migration in vitro. Blocking of PFKFB3 by selective inhibitor PFK-015 or glycolytic inhibitor 2-DG significantly restrained renal cancer cells' neoplastic potential. PFK-015 and sunitinib could synergistically inhibit pRCC cells proliferation. Glycolysis-Immune Risk Signature is closely associated with pRCC prognosis, progression, immune infiltration, and therapeutic response. PFKFB3 may serve as a pivotal glycolysis regulator and mediates Sunitinib resistance in pRCC patients.

PFKFB3 regulates breast cancer tumorigenesis and Fulvestrant sensitivity by affecting ERα stability.

Estrogen receptor alpha (ERα) is expressed in approximately 70% of breast cancer cases and determines the sensitivity and effectiveness of endocrine therapy. 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase3 (PFKFB3) is a glycolytic enzyme that is highly expressed in a great many human tumors, and recent studies have shown that it plays a significant role in improving drug sensitivity. However, the role of PFKFB3 in regulating ERα expression and the underlying mechanism remains unclear. Here, we find by using immunohistochemistry (IHC) that PFKFB3 is elevated in ER-positive breast cancer and high expression of PFKFB3 resulted in a worse prognosis. In vitro and in vivo experiments verify that PFKFB3 promotes ER-positive breast cancer cell proliferation. The overexpression of PFKFB3 promotes the estrogen-independent ER-positive breast cancer growth. In an estrogen-free condition, RNA-sequencing data from MCF7 cells treated with siPFKFB3 showed enrichment of the estrogen signaling pathway, and a luciferase assay demonstrated that knockdown of PFKFB3 inhibited the ERα transcriptional activity. Mechanistically, down-regulation of PFKFB3 promotes STUB1 binding to ERα, which accelerates ERα degradation by K48-based ubiquitin linkage. Finally, growth of ER-positive breast cancer cells in vivo was more potently inhibited by fulvestrant combined with the PFKFB3 inhibitor PFK158 than for each drug alone. In conclusion, these data suggest that PFKFB3 is identified as an adverse prognosis factor for ER-positive breast cancer and plays a previously unrecognized role in the regulation of ERα stability and activity. Our results further explores an effective approach to improve fulvestrant sensitivity through the early combination with a PFKFB3 inhibitor.

PFKFB3-mediated glycolysis in hepatic stellate cells promotes liver regeneration.

Hepatic stellate cells (HSCs) perform a significant function in liver regeneration (LR) by becoming active. We propose to investigate if activated HSCs enhance glycolysis via PFKFB3, an essential glycolytic regulator, and whether targeting this pathway could be beneficial for LR. The liver and isolated HSCs of mice subjected to 2/3 partial hepatectomy (PHx) exhibited a significant rise in PFKFB3 expression, as indicated by quantitative RT-PCR analyses and Western blotting. Also, the primary HSCs of mice subjected to PHx have a significant elevation of the glycolysis level. Knocking down PFKFB3 significantly diminished the enhancement of glycolysis by PDGF in human LX2 cells. The hepatocyte proliferation in mice treated with PHx was almost completely prevented when the PFKFB3 inhibitor 3PO was administered, emerging that PFKFB3 is essential in LR. Furthermore, there was a decline in mRNA expression of immediate early genes and proinflammatory cytokines. In terms of mechanism, both the p38 MAP kinase and ERK1/2 phosphorylation in LO2 cells and LO2 proliferation were significantly reduced by the conditioned medium (CM) obtained from LX2 cells with either PFKFB3 knockdown or inhibition. Compared to the control group, isolated hepatocytes from 3PO-treated mice showed decreased p38 MAP kinase and ERK1/2 phosphorylation and proliferation. Thus, LR after PHx involves the activation of PFKFB3 in HSCs, which enhances glycolysis and promotes lactate production, thereby facilitating hepatocyte proliferation via the p38/ERK MAPK signaling pathway.

mTORC2-AKT signaling to PFKFB2 activates glycolysis that enhances stemness and tumorigenicity of intestinal epithelial cells.

Although elevated glycolysis has been widely recognized as a hallmark for highly proliferating cells like stem cells and cancer, its regulatory mechanisms are still being updated. Here, we found a previously unappreciated mechanism of mammalian target of rapamycin complex 2 (mTORC2) in regulating glycolysis in intestinal stem cell maintenance and cancer progression. mTORC2 key subunits expression levels and its kinase activity were specifically upregulated in intestinal stem cells, mouse intestinal tumors, and human colorectal cancer (CRC) tissues. Genetic ablation of its key scaffolding protein Rictor in both mouse models and cell lines revealed that mTORC2 played an important role in promoting intestinal stem cell proliferation and self-renewal. Moreover, utilizing mouse models and organoid culture, mTORC2 loss of function was shown to impair growth of gut adenoma and tumor organoids. Based on these findings, we performed RNA-seq and noticed significant metabolic reprogramming in Rictor conditional knockout mice. Among all the pathways, carbohydrate metabolism was most profoundly altered, and further studies demonstrated that mTORC2 promoted glycolysis in intestinal epithelial cells. Most importantly, we showed that a rate-limiting enzyme in regulating glycolysis, 6-phosphofructo-2-kinase (PFKFB2), was a direct target for the mTORC2-AKT signaling. PFKFB2 was phosphorylated upon mTORC2 activation, but not mTORC1, and this process was AKT-dependent. Together, this study has identified a novel mechanism underlying mTORC2 activated glycolysis, offering potential therapeutic targets for treating CRC.

Loss of Cardiac PFKFB2 Drives Metabolic, Functional, and Electrophysiological Remodeling in the Heart.

BACKGROUND: Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) is a critical glycolytic regulator responsible for upregulation of glycolysis in response to insulin and adrenergic signaling. PFKFB2, the cardiac isoform of PFK-2, is degraded in the heart in the absence of insulin signaling, contributing to diabetes-induced cardiac metabolic inflexibility. However, previous studies have not examined how the loss of PFKFB2 affects global cardiac metabolism and function. METHODS AND RESULTS: To address this, we have generated a mouse model with a cardiomyocyte-specific knockout of PFKFB2 (cKO). Using 9-month-old cKO and control mice, we characterized the impacts of PFKFB2 on cardiac metabolism, function, and electrophysiology. cKO mice have a shortened life span of 9 months. Metabolically, cKO mice are characterized by increased glycolytic enzyme abundance and pyruvate dehydrogenase activity, as well as decreased mitochondrial abundance and beta oxidation, suggesting a shift toward glucose metabolism. This was supported by a decrease in the ratio of palmitoyl carnitine to pyruvate-dependent mitochondrial respiration in cKO relative to control animals. Metabolomic, proteomic, and Western blot data support the activation of ancillary glucose metabolism, including pentose phosphate and hexosamine biosynthesis pathways. Physiologically, cKO animals exhibited impaired systolic function and left ventricular dilation, represented by reduced fractional shortening and increased left ventricular internal diameter, respectively. This was accompanied by electrophysiological alterations including increased QT interval and other metrics of delayed ventricular conduction. CONCLUSIONS: Loss of PFKFB2 results in metabolic remodeling marked by cardiac ancillary pathway activation. This could delineate an underpinning of pathologic changes to mechanical and electrical function in the heart.

[Effect of PFKFB3 on inflammatory activation of polymorphonuclear myeloid-derived suppressor cell in acute myocardial infarction].

OBJECTIVE: To investigate the correlation between 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3) and the inflammatory activation of polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) in acute myocardial infarction (AMI), and to evaluate the effect of intervention targeting PFKFB3 on the inflammatory activation of PMN-MDSC during AMI. METHODS: (1) Clinical trial section: a observational study was conducted. The patients with acute coronary syndrome (ACS) admitted to Zhenjiang Fourth People's Hospital were enrolled, and they were divided into AMI group and non-AMI group according to clinical diagnosis. The peripheral venous blood of the two groups was collected to detect the proportion of PMN-MDSC, and the expression of PFKFB3 gene in mononuclear cells was detected by real-time quantitative polymerase chain reaction (RT-qPCR). (2) Basic experiment section: a total of 30 male C57 mice (aged 6-8 weeks) were divided into normal control group (n = 5), Sham group (n = 5), AMI model group (n = 10) and PFKFB3 inhibitor PKF-15 intervention group (n = 10) according to random number table method. The AMI model of mice was reproduced by left anterior descending coronary artery (LADCA) ligation, and the mice in the Sham group did not attach the artery after thoracotomy. The PKF-15 intervention group was intraperitoneally injected with PKF-15 (20 µg/g) at the same time of LADCA ligation. Normal control mice did not receive any treatment. Peripheral venous blood and myocardial tissue of mice were collected 24 hours after modeling. Both the circulating PMN-MDSC ratio and the infiltration of PMN-MDSC in myocardial tissue were detected. After staining with hematoxylin-eosin (HE), the degree of inflammatory damage in mouse myocardial tissue was observed under light microscopy. PMN-MDSC were isolated from mice with flow cytometry, and the gene expressions of PFKFB3 and inflammatory factors were measured by RT-qPCR. RESULTS: (1) Clinical trial section: the circulating PMN-MDSC ratio of patients in the AMI group (n = 25) was significantly higher than that in the non-AMI group [n = 20; (8.53±0.96)% vs. (1.13±0.39)%, P < 0.01], and PFKFB3 gene expression in the peripheral blood mononuclear cells was also increased (2-ΔΔCt: 1.18±0.19 vs. 0.96±0.16, P < 0.01). Pearson correlation analysis showed that circulating PMN-MDSC ratio was positively correlated with PFKFB3 gene expression in mononuclear cells in AMI patients (r = 0.608, P = 0.001). (2) Basic experimental section: the circulating PMN-MDSC ratio and the infiltration of PMN-MDSC in myocardial tissue of AMI mice were significantly higher than those in the normal control group and Sham group. PFK-15 intervention could reduce the ratio of PMN-MDSC in the peripheral blood and myocardial tissue of AMI mice [(26.33±5.27)% vs. (75.12±5.02)% in peripheral blood, (20.87±2.97)% vs. (35.28±4.36)% in myocardial tissue, both P < 0.01]. Under light microscopy, the myocardial cells in the AMI modal group were disordered and a large number of inflammatory cells infiltrated. PFK-15 intervention could maintain a normal arrangement of cardiomyocytes and reduce the infiltration of inflammatory cells. The gene expression levels of PFKFB3 in the peripheral blood and myocardial tissue as well as the inflammatory factors in the myocardial tissue of AMI mice were significantly higher than those in the normal control group and Sham group. PKF-15 intervention could effectively reduce the gene expression levels of PFKFB3 in the peripheral blood and myocardial tissue as well as the inflammatory factors in the myocardial tissue of AMI mice [PFKFB3 mRNA (2-ΔΔCt): 1.01±0.09 vs. 1.40±0.12 in peripheral blood, 0.95±0.09 vs. 1.47±0.10 in myocardial tissue; myocardial tissue tumor necrosis factor-α (TNF-α) mRNA (2-ΔΔCt) was 14.55±3.99 vs. 29.66±3.90, interleukin-1ß (IL-1ß) mRNA (2-ΔΔCt) was 8.72±1.35 vs. 18.53±2.43, IL-6 mRNA (2-ΔΔCt) was 11.87±2.97 vs. 19.82±4.32, all P < 0.01]. CONCLUSIONS: The activation of PFKFB3 is closely related to the inflammatory activation of PMN-MDSC during AMI. Inhibition of PFKFB3 activity can inhibit the inflammatory activation of PMN-MDSC and reduce myocardial inflammatory injury.

Down-Regulation of CPEB4 Alleviates Preeclampsia through the Inhibition of Ferroptosis by PFKFB3.

Gestational diabetes mellitus (GDM) complicated with preeclampsia can lead to polyhydramnios, ketosis. Herein, we explored that CPEB4 in cancer progression of preeclampsia and its underlying mechanism. All the serum samples were collected from patients with preeclampsia. These was the induction of CPEB4 in patients with preeclampsia. The serum of CPEB4 mRNA expression was positive correlation with Proteinuria, systolic blood pressure and diastolic blood pressure in patients. The serum of CPEB4 mRNA expression was also negative correlation with body weight of infant in patients. The serum of CPEB4 mRNA expression also was negative correlation with GPX4 level and GSH activity level in patients. The serum of CPEB4 mRNA expression was positive correlation with iron content in patients. CPEB4 gene inhibited trophoblast cell proliferation. CPEB4 gene promoted trophoblast cell ferroptosis by mitochondrial damage. CPEB4 gene induced PFKFB3 expression by the inhibition of PFKFB3 Ubiquitination. PFKFB3 inhibitor reduced the effects of CPEB4 on cell proliferation and ferroptosis of trophoblast cell. Taken together, the CPEB4 promoted trophoblast cell ferroptosis through mitochondrial damage by the induction of PFKFB3 expression, CPEB4 as an represents a potential therapeutic strategy for the treatment of preeclampsia or various types of GDM.

Triptolide restrains the growth, invasion, stemness, and glycolysis of non-small cell lung cancer cells by PFKFB2-mediated PI3K/AKT pathway.

Triptolide (TP) has been found to have anti-tumor effects. However, more potential molecular mechanisms of TP in the progression of non-small cell lung cancer (NSCLC) deserve further investigation. Cell proliferation, apoptosis, invasion, and stemness were detected by cell counting kit 8 assay, EdU assay, flow cytometry, transwell assay, and sphere formation assay. Cell glycolysis was evaluated by corresponding assay kits. 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2) expression was measured by western blot (WB), qRT-PCR and immunohistochemical staining. PI3K/AKT pathway-related markers were determined by WB. Besides, xenograft tumor model was conducted to evaluate the anti-tumor effect of TP in NSCLC. Our results revealed that TP treatment suppressed NSCLC cell proliferation, invasion, stemness, glycolysis, and enhanced apoptosis. PFKFB2 was upregulated in NSCLC tissues and cells, and its expression was decreased by TP. PFKFB2 knockdown restrained NSCLC cell functions, and its overexpression also eliminated TP-mediated NSCLC cell functions inhibition. TP decreased PFKFB2 expression to inactivate PI3K/AKT pathway. Moreover, PI3K/AKT pathway inhibitor LY294002 also could reverse the promoting effect of PFKFB2 on NSCLC cell functions. In addition, TP suppressed NSCLC tumorigenesis by inhibiting PFKFB2/PI3K/AKT pathway. In conclusion, TP exerted anti-tumor role in NSCLC, which was achieved by reducing PFKFB2 expression to inactivate PI3K/AKT pathway.

HP promotes neutrophil inflammatory activation by regulating PFKFB2 in the glycolytic metabolism of sepsis.

BACKGROUND: Sepsis, described as an inflammatory reaction to an infection, is a very social health problem with high mortality. This study aims to explore the new mechanism in the progression of sepsis. METHODS: We downloaded the GSE69528 dataset to screen differentially expressed genes (DEGs) for WGCNA, in which the key module was identified and analyzed by DMNC algorithm, expression verification and ROC curve analysis to identify the hub gene. Furthermore, the hub gene was analyzed by immunoassay, and the potential mechanism of hub gene in neutrophils was investigated by in vitro experiments. RESULTS: The turquoise module was the key module for sepsis in WGCNA on 94 DEGs. The top 20 genes of DMNC network were verified in GSE69528 and GSE9960, and 10 significant genes were obtained for ROC analysis. Based on the ROC curves, HP was considered the hub gene in sepsis, and its expression difference in sepsis and control groups was substantially significant. Further, it was demonstrated the knockdown of HP and PFKFB3 could suppress glycolysis and inflammatory cytokine levels in dHL-60 cell treated with LPS. CONCLUSION: In conclusion, HP is identified as a potential diagnostic indicator for sepsis patients, and HP promotes neutrophil inflammatory activation by regulating PFKFB2 in the glycolytic metabolism of sepsis confirmed by in vitro experiments. These will help us deepen the molecular mechanism of sepsis.

Targeting the SphK1/S1P/PFKFB3 axis suppresses hepatocellular carcinoma progression by disrupting glycolytic energy supply that drives tumor angiogenesis.

BACKGROUND: Hepatocellular carcinoma (HCC) remains a leading life-threatening health challenge worldwide, with pressing needs for novel therapeutic strategies. Sphingosine kinase 1 (SphK1), a well-established pro-cancer enzyme, is aberrantly overexpressed in a multitude of malignancies, including HCC. Our previous research has shown that genetic ablation of Sphk1 mitigates HCC progression in mice. Therefore, the development of PF-543, a highly selective SphK1 inhibitor, opens a new avenue for HCC treatment. However, the anti-cancer efficacy of PF-543 has not yet been investigated in primary cancer models in vivo, thereby limiting its further translation. METHODS: Building upon the identification of the active form of SphK1 as a viable therapeutic target in human HCC specimens, we assessed the capacity of PF-543 in suppressing tumor progression using a diethylnitrosamine-induced mouse model of primary HCC. We further delineated its underlying mechanisms in both HCC and endothelial cells. Key findings were validated in Sphk1 knockout mice and lentiviral-mediated SphK1 knockdown cells. RESULTS: SphK1 activity was found to be elevated in human HCC tissues. Administration of PF-543 effectively abrogated hepatic SphK1 activity and significantly suppressed HCC progression in diethylnitrosamine-treated mice. The primary mechanism of action was through the inhibition of tumor neovascularization, as PF-543 disrupted endothelial cell angiogenesis even in a pro-angiogenic milieu. Mechanistically, PF-543 induced proteasomal degradation of the critical glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3, thus restricting the energy supply essential for tumor angiogenesis. These effects of PF-543 could be reversed upon S1P supplementation in an S1P receptor-dependent manner. CONCLUSIONS: This study provides the first in vivo evidence supporting the potential of PF-543 as an effective anti-HCC agent. It also uncovers previously undescribed links between the pro-cancer, pro-angiogenic and pro-glycolytic roles of the SphK1/S1P/S1P receptor axis. Importantly, unlike conventional anti-HCC drugs that target individual pro-angiogenic drivers, PF-543 impairs the PFKFB3-dictated glycolytic energy engine that fuels tumor angiogenesis, representing a novel and potentially safer therapeutic strategy for HCC.

Drug-repurposing by virtual and experimental screening of PFKFB3 inhibitors for pancreatic cancer therapy.

Pancreatic cancer (PC) is the most frequently occurring cancer, with few effective treatments and a 5-year survival rate of only about 11%. It is characterized by stiff interstitium and pressure on blood vessels, leading to an increased glycolytic metabolism. PFKFB3 plays an important role in glycolysis, and its products (fructose-2,6-bisphosphate), which are allosteric PFK1 activators, limit the glycolytic rate. In this study, 14 PFKFB3 inhibitors were obtained by virtually screening the FDA-approved compound library. Subsequently, the in-vitro investigations confirmed that Lomitapide and Cabozantinib S-malate exhibit the excellent potential to inhibit PFKFB3. The combined administration of Lomitapide and Gemcitabine at a certain molar ratio indicated an enhanced anti-tumor effect in Orthotopic Pancreatic Cancer (OPC) models. This investigation provides a new treatment strategy for PC therapy.

A Lactic Acid Metabolism-Related Gene Signature for Predicting Clinical Outcome and Tumor Microenvironmental Status in Patients with Hepatocellular Carcinoma.

This study aims to build a prognostic model based on lactic acid metabolism-related genes (LMRGs) to predict survival outcomes and tumor microenvironment status of Hepatocellular carcinoma (HCC) patients. The model was used to calculate riskscores of clinical samples. Survival analysis and Cox regression analysis were conducted to verify the independence and reliability of the riskscore to determine its clinical significance in prognosis evaluation of HCC. Additionally, we conducted a comprehensive analysis of tumor mutation burden (TMB), immune cell infiltration, and gene set molecular function in the high- and low-risk groups. We obtained 134 LMRGs mainly involved in cellular calcium homeostasis and calcium signaling pathways. The LMRGs in the risk assessment model included PFKFB4, SLC16A3, ADRA2B, SLC22A1, QRFPR, and PROK1. This study discovered much shorter overall survival and median survival time of patients with higher riskscores when compared to those with lower riskscores. It was indicated that for independent prediction of patients' prognosis, the riskscore had a significant clinical value. A remarkable difference was also found regarding TMB between the two groups. Finally, cell experiments demonstrated that the knockout of PFKFB4 and SLC16A3 genes suppressed lactate. Our research demonstrated that the riskscore, established based on LMRGs, is a promising biomarker.

PFKFB3-Meditated Glycolysis via the Reactive Oxygen Species-Hypoxic Inducible Factor 1α Axis Contributes to Inflammation and Proliferation of Staphylococcus aureus in Epithelial Cells.

Mastitis caused by antibiotic-resistant strains of Staphylococcus aureus is a significant concern in the livestock industry due to the economic losses it incurs. Regulating immunometabolism has emerged as a promising approach for preventing bacterial inflammation. To investigate the possibility of alleviating inflammation caused by S aureus infection by regulating host glycolysis, we subjected the murine mammary epithelial cell line (EpH4-Ev) to S aureus challenge. Our study revealed that S aureus can colonize EpH4-Ev cells and promote inflammation through hypoxic inducible factor 1α (HIF1α)-driven glycolysis. Notably, the activation of HIF1α was found to be dependent on the production of reactive oxygen species (ROS). By inhibiting PFKFB3, a key regulator in the host glycolytic pathway, we successfully modulated HIF1α-triggered metabolic reprogramming by reducing ROS production in S aureus-induced mastitis. Our findings suggest that there is a high potential for the development of novel anti-inflammatory therapies that safely inhibit the glycolytic rate-limiting enzyme PFKFB3.

IL1R2 promotes retinal angiogenesis to participate in retinopathy of prematurity by activating the HIF1α/PFKFB3 pathway.

Retinopathy of prematurity (ROP) is the leading cause of blindness in children, but there is no safe and effective treatment available. Interleukin-1 receptor type 2 (IL1R2) acts as a decoy receptor for IL-1 may affect ROP progression. This study aimed to investigate the role of IL1R2 in ROP. A microglial cell model was established under hypoxia conditions and co-cultured with choroidal endothelial cells, while an oxygen-induced retinopathy (OIR) model was also established. Microglial activation and IL1R2 levels in retinal tissues were analyzed using immunofluorescence assay. Endothelial cell migration was evaluated by Transwell assay and scratch test, angiogenesis was assessed using ELISA and tube formation assay, and proliferation was evaluated by EdU assay. The HIF1α/PFKFB3 pathway was analyzed by western blot. We observed that IL1R2 expression was predicted to be upregulated in ROP and was increased in hypoxia-treated BV2 cells. Additionally, IL1R2 levels were upregulated in the retinal tissues of OIR mice and correlated with microglial activation. In vitro experiments, we found that hypoxia promoted endothelial cell migration, angiogenesis, proliferation, and activated the HIF1α/PFKFB3 pathway, which were rescued by IL1R2 knockdown. Moreover, NHWD-870 (a HIF1α/PFKFB3 pathway inhibitor) suppressed endothelial cell migration, angiogenesis, and proliferation induced by IL1R2 overexpression. In conclusion, IL1R2 facilitates the migration, angiogenesis, and proliferation of choroidal endothelial cells by activating the HIF1α/PFKFB3 pathway to regulate ROP progression.

An ERK5-PFKFB3 axis regulates glycolysis and represents a therapeutic vulnerability in pediatric diffuse midline glioma.

Metabolic reprogramming in pediatric diffuse midline glioma is driven by gene expression changes induced by the hallmark histone mutation H3K27M, which results in aberrantly permissive activation of oncogenic signaling pathways. Previous studies of diffuse midline glioma with altered H3K27 (DMG-H3K27a) have shown that the RAS pathway, specifically through its downstream kinase, extracellular-signal-related kinase 5 (ERK5), is critical for tumor growth. Further downstream effectors of ERK5 and their role in DMG-H3K27a metabolic reprogramming have not been explored. We establish that ERK5 is a critical regulator of cell proliferation and glycolysis in DMG-H3K27a. We demonstrate that ERK5 mediates glycolysis through activation of transcription factor MEF2A, which subsequently modulates expression of glycolytic enzyme PFKFB3. We show that in vitro and mouse models of DMG-H3K27a are sensitive to the loss of PFKFB3. Multi-targeted drug therapy against the ERK5-PFKFB3 axis, such as with small-molecule inhibitors, may represent a promising therapeutic approach in patients with pediatric diffuse midline glioma.

PFKFB3 promotes endometriosis cell proliferation via enhancing the protein stability of ß-catenin.

Endometriosis is a common inflammatory disease in women of reproductive age and is highly associated with infertility. However, the molecular mechanism of endometriosis remains unclear. 6-Phosphofructose-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) is a key enzyme in glycolysis and plays an important regulatory role in the development of cancer. Here we found that PFKFB3 is highly expressed in endometriotic tissues. PFKFB3 promotes the proliferation and growth of endometriosis cells. Meanwhile, PFKFB3 promotes glycolysis in endometriosis cells. Furthermore, PFKFB3 promotes migration and invasion of endometriosis cells. On this basis, we found that PFKFB3 promotes epithelial-mesenchymal transition (EMT) in endometriosis cells. PFKFB3 interacts with the essential factor of EMT, ß-catenin, and promotes the protein stability of ß-catenin. In addition, the PFKFB3 inhibitor PFK-015 inhibites the growth of endometriosis cells and the development of endometrial tissue. In conclusion, our study shows that PFKFB3 plays an important role in the development of endometriosis and provides new ideas for the clinical diagnosis or treatment of endometriosis.