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.

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.

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.

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.

Targeted metabolomics reveals PFKFB3 as a key target for elemene-mediated inhibition of glycolysis in prostate cancer cells.

BACKGROUND: Elemene, an active anticancer extract derived from Curcuma wenyujin, has well-documented anticarcinogenic properties. Nevertheless, the role of elemene in prostate cancer (PCa) and its underlying molecular mechanism remain elusive. PURPOSE: This study focuses on investigating the anti-PCa effects of elemene and its underlying mechanisms. METHODS: Cell-based assays, including CCK-8, scratch, colony formation, cell cycle, and apoptosis experiments, to comprehensively assess the impact of elemene on PCa cells (LNCaP and PC3) in vitro. Additionally, we used a xenograft model with PC3 cells in nude mice to evaluate elemene in vivo efficacy. Targeted metabolomics analysis via HILIC-MS/MS was performed to investigate elemene potential target pathways, validated through molecular biology experiments, including western blotting and gene manipulation studies. RESULTS: In this study, we discovered that elemene has remarkable anti-PCa activity in both in vitro and in vivo settings, comparable to clinical chemotherapeutic drugs but with fewer side effects. Using our established targeted metabolomics approach, we demonstrated that ß-elemene, elemene's primary component, effectively inhibits glycolysis in PCa cells by downregulating 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) expression. Furthermore, we found that ß-elemene accomplishes this downregulation by upregulating p53 and FZR1. Knockdown and overexpression experiments conclusively confirmed the pivotal role of PFKFB3 in mediating ß-elemene's anti-PCa activity. CONCLUSION: This finding presents compelling evidence that elemene exerts its anti-PCa effect by suppressing glycolysis through the downregulation of PFKFB3. This study not only improves our understanding of elemene in PCa treatment but also provides valuable insights for developing more effective and safer therapies for PCa.

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.

Inhibition of PFKFB3 Expression Stimulates Macrophage-Mediated Lymphangiogenesis Post-Acute Myocardial Infarction.

BACKGROUND: The dilation of lymphatic vessels plays a critical role in maintaining heart function, while a lack thereof could contribute to heart failure (HF), and subsequently to an acute myocardial infarction (AMI). Macrophages participate in the induction of lymphangiogenesis by secreting vascular endothelial cell growth factor C (VEGF-C), although the precise mechanism remains unclear. METHODS: Intramyocardial injections of adeno-associated viruses (AAV9) to inhibit the expression of VEGFR3 (VEGFR3 shRNA) or promote the expression of VEGFR3 (VEGFR3 ORF) in the heart; Myh6-mCherry B6 D2-tg mice and flow cytometry were used to evaluate the number of myocellular debris in the mediastinal lymph nodes; fluorescence staining and qPCR were used to evaluate fluorescence analysis; seahorse experiment was used to evaluate the level of glycolysis of macrophages; Lyz2𝐶𝑟𝑒, VEGFCfl/fl, and PFKFB3fl/fl mice were used as a model to knock out the expression of VEGF-C and PFKFB3 in macrophages. RESULTS: The escalation of VEGFR3 in cardiac tissue can facilitate the drainage of myocardial debris to the mediastinal lymph nodes, thereby improving cardiac function and reducing fibrosis after reperfusion injury. Conversely, myeloid VEGF-C deficiency displayed an increase in macrophage counts and inflammation levels following reperfusion injury. The inhibition of the critical enzyme PFKFB3 in macrophage glycolysis can stimulate the manifestation of VEGF-C in macrophages. A deficiency in myeloid PFKFB3 is associated with induced lymphangiogenesis following reperfusion injury. CONCLUSIONS: Our initial investigations suggest that the suppression of PFKFB3 expression in macrophages could potentially stimulate the production of VEGF-C in these immune cells, which in turn may facilitate lymphangiogenesis and mitigate the inflammatory effects of I/R injury.

UBC9 stabilizes PFKFB3 to promote aerobic glycolysis and proliferation of glioblastoma cells.

Cancer cells prefer to utilizing aerobic glycolysis to generate energy and anabolic metabolic intermediates for cell growth. However, whether the activities of glycolytic enzymes can be regulated by specific posttranslational modifications, such as SUMOylation, in response to oncogenic signallings, thereby promoting the Warburg effect, remain largely unclear. Here, we demonstrate that phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a key glycolytic enzyme, interacts with SUMO-conjugating enzyme UBC9 and is SUMOylated at K302 in glioblastoma cells. Expression of UBC9, which competitively prevents the binding of ubiquitin E3 ligase APC/C to PFKFB3 and subsequent PFKFB3 polyubiquitination, increases PFKFB3 stability and expression. Importantly, EGFR activation increases the interaction between UBC9 and PFKFB3, leading to increased SUMOylation and expression of PFKFB3. This increase is blocked by inhibition of EGFR-induced AKT activation whereas expression of activate AKT by itself was sufficient to recapitulate EGF-induced effect. Knockout of PFKFB3 expression decreases EGF-enhanced lactate production and GBM cell proliferation and this decrease was fully rescued by reconstituted expression of WT PFKFB3 whereas PFKFB3 K302R mutant expression abrogates EGF- and UBC9-regulated lactate production and GBM cell proliferation. These findings reveal a previously unknown mechanism underlying the regulation of the Warburg effect through the EGFR activation-induced and UBC9-mediated SUMOylation and stabilization of PFKFB3.

miR-21-5p inhibits the growth of brain glioma cells through regulating the glycolysis mediated by PFKFB2.

Brain glioma is a common gynecological tumor. MicroRNA (miRNA) plays a very important role in the pathogenesis and development of tumors. It was found that glycolysis played important regulatory roles in tumor growth. The present study aims to investigate the expression pattern of miR-21-5p in brain glioma cells. We examined miR-21-5p and PFKFB2 levels in brain glioma cells via qRT-PCR. Then we performed CCK-8 and Transwell migration assays and determined glucose uptake and lactose production to unveil the properties of miR-21-5p in invasion, cell viability, along with glycolysis in brain glioma cells. Luciferase activity assay was implemented to elucidate if PFKFB2 was a miR-21-5p target gene. Western blotting and qRT-PCR were executed to further validate that miR-21-5p targeted PFKFB2. We repeated these functional assays to observe whether miR-21-5p could impede the function of PFKFB2. qRT-PCR signified that miR-21-5p was elevated in brain glioma tissues in contrast to matching adjacent normal tissues. Functional assays disclosed that elevation of miR-21-5p promoted cell viability, invasion, together with glycolysis. Luciferase assay indicated that PFKFB2 was a miR-21-5p target gene. Moreover, miR-21-inhibit could hinder cell viability, invasion, and glycolysis triggered by overexpression of PFKFB2 in brain glioma cells. miR-21-5p level is elevated in brain glioma and can impede brain glioma cell growth via regulating the glycolysis mediated by PFKFB2, thus is a potential target of treating brain glioma.

Differential roles of highly expressed PFKFB4 in colon adenocarcinoma patients.

Colon adenocarcinoma (COAD) is a common malignant tumor, and the role of the protein PFKFB4 in glycolysis and pentose phosphate pathways is crucial. Researchers investigated the clinical significance of PFKFB4 in COAD by studying its expression in 79 tissue samples using immunohistochemistry. We found that PFKFB4 expression was significantly higher in COAD patients, particularly in the sigmoid colon. Interestingly, high PFKFB4 expression was associated with both improved overall survival (OS) and worse progression-free survival (PPS) in COAD patients. Further analysis revealed that genes associated with PFKFB4 were linked to various metabolic pathways, including amino acid biosynthesis, glycolysis, gluconeogenesis, glucose metabolism, and inflammatory response. PFKFB4 expression also showed correlations with the infiltration of different immune cell types in COAD patients, such as CD8+ T cells, CD4+ T cells, regulatory T cells (Tregs), macrophages, neutrophils, dendritic cells, active mast cells, and resting NK cells. Overall, the relationship between PFKFB4 expression and the prognosis of COAD is complex and diverse, possibly playing different roles at different stages of the disease. Moreover, its mechanism might involve interactions with various metabolic pathways and immune infiltration in the tumor microenvironment. These findings provide valuable insights into the potential role of PFKFB4 as a biomarker or therapeutic target in COAD.

Transforming growth factor ß1 upregulates 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase-4 expression in A549 and MCF-10A cells.

Transforming growth factor ß1 (TGFß1) induces a cellular process known as epithelial-mesenchymal transition (EMT) associated with metabolic reprogramming, including enhanced glycolysis. Given the involvement of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFKFB) enzymes in glycolysis, we aimed to investigate whether TGFß1 regulates expressions of PFKFB genes and if PFKFBs are required for TGFß1-driven phenotypes. A549 and MCF-10A cell lines were used as TGFß1-driven EMT models. Messenger RNA expressions of PFKFB and EMT genes were determined by real-time quantitative polymerase chain reaction. A small interfering RNA approach was used to deplete PFKFB4 expression. A Matrigel invasion assay was conducted to assess the effect of PFKFB4 silencing on the TGFß1-enhanced invasion of A549 cells. F2,6BP levels were analyzed using an enzyme-coupled assay. Glucose and lactate concentrations were determined using colorimetric assays. TGFß1 robustly induced expression of the fourth isoform of PFKFBs, PFKFB4, in both cell lines. PFKFB4 depletion partially inhibits mesenchymal transdifferentiation caused by TGFß1 in A549 cells, as assessed by microscopy. Inductions of Snail in MCF-10A cells and Fibronectin in A549 cells and repressions of E-cadherin in both cell lines by TGFß1 are attenuated by PFKFB4 silencing. PFKFB4 silencing reduces F2,6BP and glycolytic activity, although TGFß1 alone does not affect these parameters. Finally, PFKFB4 depletion suppresses the TGFß1-driven invasion of A549 cells through Matrigel. Presented data suggest that TGFß1 induces the expression of PFKFB4 in A549 and MCF-10 cells, and PFKFB4 may be required for TGFß1-driven phenotypes such as EMT and invasion in these models.

Intracellular BAPTA directly inhibits PFKFB3, thereby impeding mTORC1-driven Mcl-1 translation and killing MCL-1-addicted cancer cells.

Intracellular Ca2+ signals control several physiological and pathophysiological processes. The main tool to chelate intracellular Ca2+ is intracellular BAPTA (BAPTAi), usually introduced into cells as a membrane-permeant acetoxymethyl ester (BAPTA-AM). Previously, we demonstrated that BAPTAi enhanced apoptosis induced by venetoclax, a BCL-2 antagonist, in diffuse large B-cell lymphoma (DLBCL). This finding implied a novel interplay between intracellular Ca2+ signaling and anti-apoptotic BCL-2 function. Hence, we set out to identify the underlying mechanisms by which BAPTAi enhances cell death in B-cell cancers. In this study, we discovered that BAPTAi alone induced apoptosis in hematological cancer cell lines that were highly sensitive to S63845, an MCL-1 antagonist. BAPTAi provoked a rapid decline in MCL-1-protein levels by inhibiting mTORC1-driven Mcl-1 translation. These events were not a consequence of cell death, as BAX/BAK-deficient cancer cells exhibited similar downregulation of mTORC1 activity and MCL-1-protein levels. Next, we investigated how BAPTAi diminished mTORC1 activity and identified its ability to impair glycolysis by directly inhibiting 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) activity, a previously unknown effect of BAPTAi. Notably, these effects were also induced by a BAPTAi analog with low affinity for Ca2+. Consequently, our findings uncover PFKFB3 inhibition as an Ca2+-independent mechanism through which BAPTAi impairs cellular metabolism and ultimately compromises the survival of MCL-1-dependent cancer cells. These findings hold two important implications. Firstly, the direct inhibition of PFKFB3 emerges as a key regulator of mTORC1 activity and a promising target in MCL-1-dependent cancers. Secondly, cellular effects caused by BAPTAi are not necessarily related to Ca2+ signaling. Our data support the need for a reassessment of the role of Ca2+ in cellular processes when findings were based on the use of BAPTAi.

cGAS-STING signaling pathway promotes hypoxia-induced renal fibrosis by regulating PFKFB3-mediated glycolysis.

Hypoxia has long been considered to play an active role in the progression of fibrosis in chronic kidney disease, but its specific mechanism is not fully understood. The stimulator of interferon genes (STING) has been a research hotspot in the fields of tumor, immunity, and infection in recent years, and its role in immune and inflammatory responses related to kidney disease has gradually attracted attention. This study mainly explores the role and mechanism of STING in hypoxia-related renal fibrosis. To address this issue, we stimulated human proximal tubular epithelial (HK-2) cells with hypoxia for 48 h to construct cell models. Meanwhile, C57BL/6J male mice were used to establish a renal fibrosis model induced by renal ischemia-reperfusion injury (IRI). In our present study, we found that the GMP-AMP synthase (cGAS)-STING signaling pathway can promote the progression of renal fibrosis after hypoxic exposure, and this effect is closely related to 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3)-mediated glycolysis. Furthermore, inhibition of both STING and its downstream interferon regulatory factor 3 (IRF3) reversed elevated PFKFB3 expression, thereby attenuating hypoxia-induced renal fibrosis. Taken together, our data suggest that the cGAS-STING-IRF3-PFKFB3 signaling pathway activated under hypoxia may provide new ideas and targets for the treatment of early renal fibrosis.

THOC3 interacts with YBX1 to promote lung squamous cell carcinoma progression through PFKFB4 mRNA modification.

The THO complex (THOC) is ubiquitously involved in RNA modification and various THOC proteins have been reported to regulate tumor development. However, the role of THOC3 in lung cancer remains unknown. In this study, we identified that THOC3 was highly expressed in lung squamous cell carcinoma (LUSC) and negatively associated with prognosis. THOC3 knockdown inhibited LUSC cell growth, migration, and glycolysis. THOC3 expression was regulated by TRiC proteins, such as CCT8 and CCT6A, which supported protein folding. Furthermore, THOC3 could form a complex with YBX1 to promote PFKFB4 transcription. THOC3 was responsible for exporting PFKFB4 mRNA to the cytoplasm, while YBX1 ensured the stability of PFKFB4 mRNA by recognizing m5C sites in its 3'UTR. Downregulation of PFKFB4 suppressed the biological activities of LUSC. Collectively, these findings suggest that THOC3, folded by CCT proteins can collaborate with YBX1 to maintain PFKFB4 expression and facilitate LUSC development. Therefore, THOC3 could be considered as a novel promising therapeutic target for LUSC.

Combination of 3PO analog PFK15 and siPFKL efficiently suppresses the migration, colony formation ability, and PFK-1 activity of triple-negative breast cancers by reducing the glycolysis.

Among all the subtypes of breast cancer, triple-negative breast cancer (TNBC) has been associated with the worst prognosis. Recently, for many solid tumors (including breast cancer) metabolic reprogramming has appeared as a cancer cell hallmark, and the elevated glycolytic pathway has been linked to their aggressive phenotype. In the present study, we evaluated the prognostic and therapeutic relevance of PFKFB3 (6-phosphofructo-2- kinase/fructose-2,6-bisphosphatase) in TNBCs. Prognostic significance of PFKFB3 expression was evaluated in overall breast cancers as well as in TNBCs. PFKFB3 inhibitor (3PO potent analogue i.e., PFK15) cytotoxicity in TNBC cell lines (MDA-MB-231 and MDA-MB-468) was analyzed using an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Cancer cell physiological characteristics like clonogenicity and migration were also investigated after PFK15 treatment. As fructose-2,6-bisphosphate (F-2,6-BP), has been associated with increased PFK-1 activity, the effect of PFKFB3 inhibition by PFK15 was investigated on two major isoforms of phosphofructokinase-1 (PFK-1) in breast cancer, that is, phosphofructokinase-platelet type (PFKP) and phosphofructokinase-liver type (PFKL) (relevant to breast cancer). For PFKL inhibition, the siRNA approach was used. PFKFB3 expression was significantly correlated with inferior overall survival in breast cancer patients including TNBCs. PFK15 treatment in TNBC cells (i.e., MDA-MB-231 and MDA-MB-468) resulted in a decreased PFKP expression, thereby leading to reduced colony formation ability, migration rate, and extracellular lactate levels. However, to our surprise PFK15 treatment in both TNBC cells also resulted in elevated PFKL levels. Our results demonstrated that the combinatorial inhibition of PFK15 with siPFKL was more effective in TNBC cells, as it led to a decrease in colony formation ability, migration rate, extracellular lactate levels, and PFK-1 activity when compared with individual treatments. Using bona fide PFKFB3 inhibitor, that is, AZ67, we further show that AZ67 treatment to TNBC cells has no effect either on the expression of PFKP and PFKL, or on the lactate production. In summary, our present in vitro study demonstrated that 3PO derived PFK15 mechanism of action is totally different from AZ67 in TNBC cells. However, we advocate that the PFK15-mediated inhibition (along with PFKL) on the TNBCs migration, colony formation, and PFK-1 activity can be further explored for the therapeutic advantage of TNBC patients.

FOXK2 regulates PFKFB3 in promoting glycolysis and tumorigenesis in multiple myeloma.

Forkhead box K2 (FOXK2) is a transcription factor involved in regulating the pathophysiological processes in many types of cancers. Functioning as either an oncogene or tumor suppressor, FOXK2 is involved in cell proliferation, metastasis, DNA damage, metabolism, and autophagy. However, the functions of FOXK2 in multiple myeloma (MM) are still unexplored. Here we show that FOXK2 silencing by small interfering RNA (siRNA) prevented the expression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) via dephosphorylation of an AMP-activated protein kinase (AMPK). Consistently, suppression of FOXK2 inhibited glycolysis and cell proliferation in MM cells. Furthermore, the correlation between FOXK2 expression and disease progression in MM was evaluated using the TCGA (The Cancer Genome Atlas) database. Taken together, we identified a novel FOXK2-dependent signaling pathway involved in the regulation of PFKFB3 expression in response to glycolysis, which might serve as a potential therapeutic target in MM.

PFKFB2 is a favorable prognostic biomarker for colorectal cancer by suppressing metastasis and tumor glycolysis.

PURPOSE: This study was to investigate the biological effect of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2) in colorectal cancer (CRC). METHODS: PFKFB2 was selected by metabolism polymerase chain reaction (PCR) array from CRC cells under alkaline culture medium (pH 7.4) and acidic culture medium (pH 6.8). The expression of PFKFB2 mRNA and protein was detected by quantitative real-time PCR and immunohistochemistry in 70 paired fresh and 268 paired paraffin-embedded human CRC tissues, respectively, and then the prognostic value of PFKFB2 was investigated. The effects of PFKFB2 on CRC cells were also verified in vitro, which were through detecting the change of migration, invasion, sphere formation, proliferation, colony formation, and extracellular acidification rate of CRC cells after PFKFB2 knockdown in alkaline culture medium (pH 7.4) and overexpression in acidic culture medium (pH 6.8). RESULTS: PFKFB2 expression was downregulated in acidic culture medium (pH 6.8). In addition, we found PFKFB2 expression decreased in human CRC tissues compared with the adjacent normal tissues. Furthermore, the OS and DFS rate of CRC patients with low PFKFB2 expression was significantly shorter than those of patients with high PFKFB2 expression. Multivariate analysis indicated that low PFKFB2 expression was an independent prognostic factor for both OS and DFS in CRC patients. Moreover, the abilities of migration, invasion, spheroidizing ability, proliferation, and colony formation of CRC cells were significantly increased after depletion of PFKFB2 in alkaline culture medium (pH 7.4) and decreased after overexpression of PFKFB2 in acidic culture medium (pH 6.8) in vitro. Epithelial-mesenchymal transition (EMT) pathway was found and verified involved in the PFKFB2-mediated regulation of metastatic function in CRC cells. Further, glycolysis of CRC cells was significantly elevated after knockdown of PFKFB2 in alkaline culture medium (pH 7.4) and decreased after overexpression of PFKFB2 in acidic culture medium (pH 6.8). CONCLUSION: PFKFB2 expression is downregulated in CRC tissues and associated with worse survival for CRC patients. PFKFB2 could inhibit metastasis and the malignant progression of CRC cells by suppressing EMT and glycolysis.

Glycolytic regulatory enzyme PFKFB3 as a prognostic and tumor microenvironment biomarker in human cancers.

The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFK-2/FBPase-2, PFKFB3) is a glycolysis regulatory enzyme and plays a key role in oncogenesis of several cancers. However, the systematic study of crosstalk between PFKFB3 and Tumor microenvironment (TME) in pan-cancer has less been examined. In this study, we conducted a comprehensive analysis of the relationship between PFKFB3 expression, patient prognostic, Tumor mutational burden (TMB), Microsatellite instability (MSI), DNA mismatch repair (MMR), and especially TME, including immune infiltration, immune regulator, and immune checkpoint, across 33 types of tumors using datasets of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). We found that PFKFB3 expression was significantly correlated with patient prognostic and TME factors in various tumors. Moreover, we confirmed that PFKFB3 was an independent prognostic factor for kidney renal papillary cell carcinoma (KIRP), and established a risk prognostic model based on the expression of PFKFB3 as a clinical risk factor, which has a good predictive ability. Our study indicated that PFKFB3 is a potent regulatory factor for TME and has the potential to be a valuable prognostic biomarker in human tumor therapy.

Reprogramming of glucose metabolism via PFKFB4 is critical in FGF16-driven invasion of breast cancer cells.

Fibroblast growth factors (FGFs) are expressed in both developing and adult tissues and play important roles in embryogenesis, tissue homeostasis, angiogenesis, and neoplastic transformation. Here, we report the elevated expression of FGF16 in human breast tumor and investigate its potential involvement in breast cancer progression. The onset of epithelial-mesenchymal transition (EMT), a prerequisite for cancer metastasis, was observed in human mammary epithelial cell-line MCF10A by FGF16. Further study unveiled that FGF16 alters mRNA expression of a set of extracellular matrix genes to promote cellular invasion. Cancer cells undergoing EMT often show metabolic alteration to sustain their continuous proliferation and energy-intensive migration. Similarly, FGF16 induced a significant metabolic shift toward aerobic glycolysis. At the molecular level, FGF16 enhanced GLUT3 expression to facilitate glucose transport into cells, which through aerobic glycolysis generates lactate. The bi-functional protein, 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 4 (PFKFB4) was found to be a mediator in FGF16-driven glycolysis and subsequent invasion. Furthermore, PFKFB4 was found to play a critical role in promoting lactate-induced cell invasion since silencing PFKFB4 decreased lactate level and rendered the cells less invasive. These findings support potential clinical intervention of any of the members of FGF16-GLUT3-PFKFB4 axis to control the invasion of breast cancer cells.