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Glycolysis is a crucial metabolic process in rapidly proliferating cells such as cancer cells. Phosphofructokinase-1 (PFK-1) is a key rate-limiting enzyme of glycolysis. Its efficiency is allosterically regulated by numerous substances occurring in the cytoplasm. However, the most potent regulator of PFK-1 is fructose-2,6-bisphosphate (F-2,6-BP), the level of which is strongly associated with 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase activity (PFK-2/FBPase-2, PFKFB). PFK-2/FBPase-2 is a bifunctional enzyme responsible for F-2,6-BP synthesis and degradation. Four isozymes of PFKFB (PFKFB1, PFKFB2, PFKFB3, and PFKFB4) have been identified. Alterations in the levels of all PFK-2/FBPase-2 isozymes have been reported in different diseases. However, most recent studies have focused on an increased expression of PFKFB3 and PFKFB4 in cancer tissues and their role in carcinogenesis. In this review, we summarize our current knowledge on all PFKFB genes and protein structures, and emphasize important differences between the isoenzymes, which likely affect their kinase/phosphatase activities. The main focus is on the latest reports in this field of cancer research, and in particular the impact of PFKFB3 and PFKFB4 on tumor progression, metastasis, angiogenesis, and autophagy. We also present the most recent achievements in the development of new drugs targeting these isozymes. Finally, we discuss potential combination therapies using PFKFB3 inhibitors, which may represent important future cancer treatment options.

Background Infantile hemangioma (IH) is the most common tumor among infants, but the exact pathogenesis of IH is largely unknown. Our previous study revealed that glucose metabolism may play an important role in the pathogenesis of IH and that the inhibition of the glycolytic key enzyme phosphofructokinase-1 suppresses angiogenesis in IH. 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) is a metabolic enzyme that converts fructose-6-bisphosphate to fructose-2,6-bisphosphate (F-2,6-BP), which is the most potent allosteric activator of the rate-limiting enzyme phosphofructokinase-1. This study was performed to explore the role of PFKFB3 in IH. Methods Microarray analysis was performed to screen the differentially expressed genes (DEGs) between proliferating and involuting IH tissues. PFKFB3 expression was examined by western blot and immunohistochemistry analyses. Cell migration, apoptosis and tube formation were analyzed. Metabolic analyses were performed to investigate the effect of PFKFB3 inhibition by PFK15. Mouse models were established to examine the effect of PFKFB3 inhibition in vivo. Results PFKFB3 was identified as one of the most significant DEGs and was more highly expressed in proliferating IH tissues and hemangioma-derived endothelial cells (HemECs) than in involuting IH tissues and human umbilical vein endothelial cells, respectively. PFKFB3 inhibition by PFK15 suppressed HemEC glucose metabolism mainly by affecting glycolytic metabolite metabolism and decreasing the glycolytic flux. Moreover, PFK15 inhibited HemEC angiogenesis and migration and induced apoptosis via activation of the apoptosis pathway. Treatment with the combination of PFK15 with propranolol had a synergistic inhibitory effect on HemECs. Moreover, PFKFB3 knockdown markedly suppressed HemEC angiogenesis. Mechanistically, inhibition of PFKFB3 suppressed the PI3K-Akt signaling pathway and induced apoptotic cell death. More importantly, the suppression of PFKFB3 by PFK15 or shPFKFB3 led to markedly reduced tumor growth in vivo. Conclusions Our findings suggest that PFKFB3 inhibition can suppress IH angiogenesis and induce apoptosis. Thus, targeting PFKFB3 may be a novel therapeutic strategy for IH.

Abstract Diabetic retinopathy (DR) is the leading cause of blindness for adults in developed countries. Both microvasculopathy and neurodegeneration are implicated in mechanisms of DR development, with neuronal impairment preceding microvascular abnormalities, which is often underappreciated in the clinic. Most current therapeutic strategies, including anti-vascular endothelial growth factor (anti-VEGF)-antibodies, aim at treating the advanced stages (diabetic macular edema and proliferative diabetic retinopathy) and fail to target the neuronal deterioration. Hence, new therapeutic approach(es) intended to address both vascular and neuronal impairment are urgently needed. The hypoxia-inducible factor 1α (HIF1α)–6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) pathway is critically implicated in the islet pathology of diabetes. Recent evidence highlighted the pathway relevance for pathologic angiogenesis and neurodegeneration, two key aspects in DR. PFKFB3 is key to the sprouting angiogenesis, along with VEGF, by determining the endothelial tip-cell competition. Also, PFKFB3-driven glycolysis compromises the antioxidative capacity of neurons leading to neuronal loss and reactive gliosis. Therefore, the HIF1α-PFKFB3 signaling pathway is unique as being a pervasive pathological component across multiple cell types in the retina in the early as well as late stages of DR. A metabolic point-of-intervention based on HIF1α-PFKFB3 targeting thus deserves further consideration in DR.

Metabolic reprogramming plays a critical role in many diseases. A recent study revealed that aerobic glycolysis in lung tissue is closely related to pulmonary fibrosis, and also occurs during lipopolysaccharide (LPS)-induced sepsis. However, whether LPS induces aerobic glycolysis in lung fibroblasts remains unknown. The present study demonstrated that LPS promotes collagen synthesis in the lung fibroblasts through aerobic glycolysis via the activation of the PI3K-Akt-mTOR/PFKFB3 pathway. Challenging the human lung fibroblast MRC-5 cell line with LPS activated the PI3K-Akt-mTOR pathway, significantly upregulated the expression of 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3), enhanced the aerobic glycolysis, and promoted collagen synthesis. These phenomena could be reversed by the PI3K-Akt inhibitor LY294002, mTOR inhibitor rapamycin, PFKFB3 inhibitor 3PO, or PFKFB3 silencing by specific shRNA, or aerobic glycolysis inhibitor 2-DG. In addition, PFKFB3 expression and aerobic glycolysis were also detected in the mouse model of LPS-induced pulmonary fibrosis, which could be reversed by the intraperitoneal injection of PFKFB3 inhibitor 3PO. Taken together, this study revealed that in LPS-induced pulmonary fibrosis, LPS might mediate lung fibroblast aerobic glycolysis through the activation of the PI3K-Akt-mTOR/PFKFB3 pathway. Lipopolysaccharide (LPS) is a vital promoter of sepsis-related pulmonary fibrosis. This study revealed that in LPS-induced pulmonary fibrosis, LPS mediates lung fibroblast aerobic glycolysis through the activation of the PI3K-Akt-mTOR/PFKFB3 pathway. Targeting the initiation of aerobic glycolysis may be a constructive treatment for pulmonary fibrosis.

Increased glycolysis in the lung vasculature has been connected to the development of pulmonary hypertension (PH). We therefore investigated whether glycolytic regulator 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (PFKFB3)-mediated endothelial glycolysis plays a critical role in the development of PH. Heterozygous global deficiency of Pfkfb3 protected mice from developing hypoxia-induced PH, and administration of the PFKFB3 inhibitor 3PO almost completely prevented PH in rats treated with Sugen 5416/hypoxia, indicating a causative role of PFKFB3 in the development of PH. Immunostaining of lung sections and Western blot with isolated lung endothelial cells showed a dramatic increase in PFKFB3 expression and activity in pulmonary endothelial cells of rodents and humans with PH. We generated mice that were constitutively or inducibly deficient in endothelial Pfkfb3 and found that these mice were incapable of developing PH or showed slowed PH progression. Compared with control mice, endothelial Pfkfb3-knockout mice exhibited less severity of vascular smooth muscle cell proliferation, endothelial inflammation, and leukocyte recruitment in the lungs. In the absence of PFKFB3, lung endothelial cells from rodents and humans with PH produced lower levels of growth factors (such as PDGFB and FGF2) and proinflammatory factors (such as CXCL12 and IL1β). This is mechanistically linked to decreased levels of HIF2A in lung ECs following PFKFB3 knockdown. Taken together, these results suggest that targeting PFKFB3 is a promising strategy for the treatment of PH.

Tumor cells often reprogram their metabolism for rapid proliferation. The roles of long noncoding RNAs (lncRNAs) in metabolism remodeling and the underlying mechanisms remain elusive. Through screening, we found that the lncRNA Actin Gamma 1 Pseudogene ( AGPG ) is required for increased glycolysis activity and cell proliferation in esophageal squamous cell carcinoma (ESCC). Mechanistically, AGPG binds to and stabilizes 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). By preventing APC/C-mediated ubiquitination, AGPG protects PFKFB3 from proteasomal degradation, leading to the accumulation of PFKFB3 in cancer cells, which subsequently activates glycolytic flux and promotes cell cycle progression. AGPG is also a transcriptional target of p53; loss or mutation of TP53 triggers the marked upregulation of AGPG . Notably, inhibiting AGPG dramatically impaired tumor growth in patient-derived xenograft (PDX) models. Clinically, AGPG is highly expressed in many cancers, and high AGPG expression levels are correlated with poor prognosis, suggesting that AGPG is a potential biomarker and cancer therapeutic target. PFKFB3 enhances glycolysis to promote cancer cell proliferation. Here, the authors identify a long noncoding RNA in esophageal squamous cell carcinoma, AGPG , which interacts with PFKFB3 and promotes its stability, leading to increased glycolysis and proliferation.

Breast cancer stem cells (BCSCs) are unique in their ability to undergo unlimited self-renewal, an essential process in breast cancer recurrence following metastatic dormancy. Emergent metastatic lesions were subjected to microarray analysis, which identified 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (Pfkfb3) as a differentially expressed gene coupled to metastatic recurrence. Here, we report that elevated Pfkfb3 expression correlates with the appearance of aggressive breast cancers and reduces relapse-free survival, as well as enhances BCSC self-renewal and metastatic outgrowth. We observe an inverse relationship between Pfkfb3 expression and autophagy, which reduces Pfkfb3 expression and elicits cellular dormancy. Targeted depletion of Atg3, Atg7, or p62/sequestosome-1 to inactivate autophagy restores aberrant Pfkfb3 expression in dormant BCSCs, leading to their reactivation of proliferative programs and outgrowth. Moreover, Pfkfb3 interacts physically with autophagy machinery, specifically the UBA domain of p62/sequestosome-1. Importantly, disrupting autophagy and this event enables Pfkfb3 to drive dormant BCSCs and metastatic lesions to recur. Cancer cells can use autophagy as a mechanism of surviving from external stresses. Here, the authors show that Pfkfb3, a glycolytic gene, is expressed in metastatic breast cancer cells but not in dormant cells that demonstrate features of autophagy.

Rheumatoid arthritis (RA) is a classic autoimmune disease characterized by uncontrolled synovial proliferation, pannus formation, cartilage injury, and bone destruction. The specific pathogenesis of RA, a chronic inflammatory disease, remains unclear. However, both key glycolysis rate-limiting enzymes, hexokinase-II (HK-II), phosphofructokinase-1 (PFK-1), and pyruvate kinase M2 (PKM2), as well as indirect rate-limiting enzymes, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), are thought to participate in the pathogenesis of RA. In here, we review the latest literature on the pathogenesis of RA, introduce the pathophysiological characteristics of HK-II, PFK-1/PFKFB3, and PKM2 and their expression characteristics in this autoimmune disease, and systematically assess the association between the glycolytic rate-limiting enzymes and RA from a molecular level. Moreover, we highlight HK-II, PFK-1/PFKFB3, and PKM2 as potential targets for the clinical treatment of RA. There is great potential to develop new anti-rheumatic therapies through safe inhibition or overexpression of glycolysis rate-limiting enzymes.

A bstract Enhanced glycolysis in cancer cells has been linked to cell protection from DNA damaging signals, although the mechanism is largely unknown. The 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) catalyzes the generation of fructose-2,6-bisphosphate, a potent allosteric stimulator of glycolysis. Intriguingly, among the four members of PFKFB family, PFKFB3 is uniquely localized in the nucleus, although the reason remains unclear. Here we show that chemotherapeutic agent cisplatin promotes glycolysis, which is suppressed by PFKFB3 deletion. Mechanistically, cisplatin induces PFKFB3 acetylation at lysine 472 (K472), which impairs activity of the nuclear localization signal (NLS) and accumulates PFKFB3 in the cytoplasm. Cytoplasmic accumulation of PFKFB3 facilitates its phosphorylation by AMPK, leading to PFKFB3 activation and enhanced glycolysis. Inhibition of PFKFB3 sensitizes tumor to cisplatin treatment in a xenograft model. Our findings reveal a mechanism for cells to stimulate glycolysis to protect from DNA damage and potentially suggest a therapeutic strategy to sensitize tumor cells to genotoxic agents by targeting PFKFB3. Enhanced glycolysis in cancer cells has been associated with protection from DNA damage. Here the authors show that DNA damaging signals induce acetylation of PFKFB3 at lysine K472 and promote its cytosolic accumulation, which enhances glycolysis, resulting in protection from cisplatin-induced cell death.

PFKFB3 (6-phosphofructo-2-kinase) is the rate-limiting enzyme of glycolysis and is overexpressed in several human cancers that are associated with poor prognosis. High PFKFB3 expression in cancer stem cells promotes glycolysis and survival in the tumor microenvironment. Inhibition of PFKFB3 by the glycolytic inhibitor PFK158 and by shRNA stable knockdown in small cell lung carcinoma (SCLC) cell lines inhibited glycolysis, proliferation, spheroid formation, and the expression of cancer stem cell markers CD133, Aldh1, CD44, Sox2, and ABCG2. These factors are also associated with chemotherapy resistance. We found that PFK158 treatment and PFKFB3 knockdown enhanced the ABCG2-interacting drugs doxorubicin, etoposide, and 5-fluorouracil in reducing cell viability under conditions of enriched cancer stem cells (CSC). Additionally, PFKFB3 inhibition attenuated the invasion/migration of SCLC cells by downregulating YAP/TAZ signaling while increasing pLATS1 via activation of pMST1 and NF2 and by reducing the mesenchymal protein expression. PFKFB3 knockdown and PFK158 treatment in a H1048 SCLC cancer stem cell-enriched mouse xenograft model showed significant reduction in tumor growth and weight with reduced expression of cancer stem cell markers, ABCG2, and YAP/TAZ. Our findings identify that PFKFB3 is a novel target to regulate cancer stem cells and its associated therapeutic resistance markers YAP/TAZ and ABCG2 in SCLC models.