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.

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.

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.

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.

Identification of PFKFB2 as a key gene for the transition from acute to old myocardial infarction in peripheral blood.

Objective: This study aims to analyze the gene expression profile of peripheral blood in different stages of myocardial infarction (MI) by transcriptome sequencing, and to study the gene expression characteristics of peripheral blood after MI. Methods: Differentially expressed genes (DEGs) and weighted gene co-expression network analysis (WGCNA) were used to identify genes and modules associated with old myocardial infarction (OMI). Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation were applied to analyze the potential functions of genes. Hub genes were identified by Random Forest Classifier. CIBERSORT was used to provide an estimate of the abundance of 22 immune cells in peripheral blood. Quantitative polymerase chain reaction (qPCR) was used to detect gene expression levels in clinical samples. The cellular components (CC) of peripheral blood were counted by an automatic hematology analyzer. Results: Through differential gene analysis and co-expression network analysis, 11 candidate genes were obtained. A random forest classifier identified 10 hub genes. Immune cell distribution of peripheral blood was found that T cell CD4 memory resting, NK cells resting, Dendritic cells activated, Mast cells resting, Monocytes and Neutrophils were correlated with OMI. Spearman correlation analysis found that PFKFB2 is related to the above immune cells. Low expression of PFKFB2 in peripheral blood of OMI was detected in clinical samples, and the relationship between PFKFB2 and peripheral blood immune cell counts was analyzed, which showed monocytes were associated with PFKFB2 in our study. Conclusion: PFKFB2 was low expressed in OMI, and related to the distribution of immune cells. PFKFB2 may play a key role in reflecting the transition from AMI to OMI, and predicting the distribution of immune cells, which provided a new perspective for improving myocardial fibrosis and adverse remodeling.

Long intergenic non-protein coding RNA 115 (LINC00115) aggravates retinoblastoma progression by targeting microRNA miR-489-3p that downregulates 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2).

Long non-coding RNAs (lncRNAs) are key regulators of cancer. However, the role of long intergenic non-protein coding RNA 115 (LINC00115) in the regulation of retinoblastoma (RB) has not yet been studied. The expression levels of LINC00115, microRNA (miR)-489-3p, and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2) in RB tissues or cells were detected by quantitative reverse transcription-polymerase chain reaction. The proliferation and migration of cells were detected by the cell counting kit-8 and Transwell assays. Luciferase reporter gene analysis and RNA immunoprecipitation assay were used to validate the target gene interactions predicted by starBase. A xenograft tumor experiment was conducted to validate the in vivo outcomes. The expression levels of LINC00115 and PFKFB2 in RB tissues were higher than those in normal tissues, while miR-489-3p showed the opposite trend. Silencing of LINC00115 inhibited the proliferation and migration of SO-RB50 and HXO-RB44 cells. An inhibitory or facilitated effect on RB tumorigenesis was observed following PFKFB2 silencing or miR-489-3p overexpression, respectively. Moreover, LINC00115 aggravated RB progression by targeting miR-489-3p, which downregulated PFKFB2. This finding improves our understanding of the relationship between LINC00115 and RB. Furthermore, miR-489-3p and PFKFB2 may be used as potential targets for RB prevention and treatment.

Knockdown circZNF131 Inhibits Cell Progression and Glycolysis in Gastric Cancer Through miR-186-5p/PFKFB2 Axis.

Gastric cancer (GC) is a prevalent and heterogeneous malignancy in the digestive system. Increasing studies have suggested that circular RNAs are implicated in GC pathogenesis. This study aimed to explore the biological role and underlying mechanism of circRNA zinc finger protein 131 (circZNF131) in GC. The expression pattern of circZNF131, microRNA-186-5p (miR-186-5p), and 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 2 (PFKFB2) mRNA in GC tissues and cells was detected by quantitative real-time polymerase chain reaction. The stability of circZNF131 was verified using ribonuclease R assay. Functional experiments were performed by colony formation assay for cloning ability analysis, transwell assay and wounding healing assay for cell metastasis, and flow cytometry for cell apoptosis. Glycolysis metabolism was investigated by determining the levels of glucose uptake and lactate production. The protein detection of apoptosis- or glycolysis-associated markers, PFKFB2, and Ki-67 was implemented by western blot or immunohistochemistry. Dual-luciferase reporter assay was conducted to identify the interaction between miR-186-5p and circZNF131 or PFKFB2. The role of circZNF131 on tumor growth in nude mice was investigated via xenograft tumor assay. Expression analysis indicated that circZNF131 was upregulated in GC tissues and cells in a stable structure. Functional analyses showed that circZNF131 knockdown suppressed GC cell colony formation ability, migration, invasion and glycolysis metabolism, and induced cell apoptosis. Mechanically, miR-186-5p was a target of circZNF131, and miR-186-5p could bind to PFKFB2. Rescue experiments presented that miR-186-5p inhibition reversed the effects of circZNF131 knockdown on GC cell growth and glycolysis, and PFKFB2 overexpression abolished the impacts of miR-186-5p restoration on GC cell progression. Moreover, circZNF131 could positively modulate PFKFB2 expression via sponging miR-186-5p. In vivo, circZNF131 knockdown hindered GC tumor growth by regulating the miR-186-5p/PFKFB2 axis. circZNF131 could exert an oncogenic role in GC malignant development through the miR-186-5p/PFKFB2 axis, which might provide novel targets for GC treatment.

miR-1297 Suppresses Osteosarcoma Proliferation and Aerobic Glycolysis by Regulating PFKFB2.

BACKGROUND: MiR-1297 is reported to function as a tumor suppressor of various cancers. However, the role of miR-1297 in the development of osteosarcoma (OS) has not been elaborated. The purpose of this study was to investigate the functional effects of miR-1297 on OS progression and the underlying mechanism. METHODS: The expression of protein and mRNA in OS cells was evaluated by Western blotting and quantitative real-time polymerase chain reaction. Cellular proliferation was investigated by cell counting kit-8, colony formation and apoptosis assays. Bioinformatics methods were used to predict target genes. The relationship between PFKFB2 and miR-1297 was demonstrated by dual-luciferase reporter assay. Metabolic changes in OS cells were monitored using an XF96 metabolic flux analyzer. RESULTS: We found that miR-1297 was downregulated in OS and that lower expression of miR-1297 promoted proliferation and contributed to the Warburg effect in OS cells. Furthermore, we showed that silencing PFKFB2 inhibited proliferation and reduced aerobic glycolysis while overexpression of PFKFB2 reduced the anti-tumor function of miR-1297 in OS cells. Mechanistically, miR-1297 acted as a tumor suppressor in OS and reduced the expression of PFKFB2 by directly targeting its 3'UTR. CONCLUSION: The miR-1297/PFKFB2 axis regulated OS proliferation by controlling the Warburg effect. Our results revealed a previously undiscovered function of miR-1297 in OS, which strongly linked metabolic alterations with cancer progression. Targeting miR-1297 may become a promising therapeutic approach for OS.

CircFLNA Acts as a Sponge of miR-646 to Facilitate the Proliferation, Metastasis, Glycolysis, and Apoptosis Inhibition of Gastric Cancer by Targeting PFKFB2.

BACKGROUND: Many studies have confirmed that circular (circRNA) is involved in the development of gastric cancer (GC). However, the role of circFLNA in the progression of GC remains unclear. METHODS: Quantitative real-time PCR (qRT-PCR) was used to measure the relative expression of circFLNA, microRNA (miR)-646 and 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 2 (PFKFB2). Cell counting kit 8 (CCK8) assay, transwell assay and flow cytometry were performed to determine the proliferation, migration, invasion and apoptosis of cells, respectively. GC tumor xenograft models were built to confirm the function of circFLNA silencing on GC tumor growth in vivo. Furthermore, the lactate production, glucose consumption, ATP level and glucose uptake were detected to assess the glycolysis of cells. Then, the interaction between miR-646 and circFLNA or PFKFB2 was confirmed using dual-luciferase reporter assay. RNA immunoprecipitation (RIP) assay was used to verify the interaction between miR-646 and circFLNA further. In addition, Western blot (WB) analysis was employed to detect the relative protein expression of PFKFB2. RESULTS: Our results found that circFLNA was upregulated in GC tissues and cells. Silencing of circFLNA could suppress the proliferation, migration, invasion, glycolysis, and enhance the apoptosis of GC cells. Also, circFLNA knockdown reduced GC tumor volume and weight in vivo. Further experiments revealed that circFLNA could sponge miR-646, and miR-646 could target PFKFB2. The rescue experiments indicated that miR-646 inhibitor could reverse the suppressive effect of circFLNA silencing on GC progression, and PFKFB2 overexpression also could invert the inhibition effect of miR-646 on GC progression. CONCLUSION: Our data concluded that circFLNA played a pro-cancer role in GC, which suggested that circFLNA might be a potential biomarker for GC treatment.

Low glucose enhanced metformin's inhibitory effect on pancreatic cancer cells by suppressing glycolysis and inducing energy stress via up-regulation of miR-210-5p.

To explore mechanisms underlying the discrepancy in anti-tumor effects of metformin on pancreatic cancer cells PANC-1 under different glucose conditions. We cultured PANC-1 cells in 25 mM and 5 mM glucose media, then treated with or without metformin. It showed that metformin significantly inhibited proliferation and viability, induced apoptosis of PANC-1 cells, which was more pronounced in low-glucose than in high-glucose group. Metformin up-regulated the expression of miR-210-5p in low glucose, but not in high glucose. miR-210-5p mimic inhibited the viability of PANC-1 cells and further enhanced the inhibitory effect of metformin. miR-210-5p down-regulated the expression of PFKFB2, a predicted target gene of miR-210-5p, reduced the activity of PFK1 and LDH. Metformin significantly inhibited the expression of phosphorylation-PFKFB2(p-PFKFB2) in the low-glucose group and inhibited the LDH activity both in the low and high glucose groups, thus inhibiting anaerobic glycolysis and inducing energy stress. Cells in the high glucose group could make a compensatory adaptation to the energy stress induced by metformin through increasing glucose consumption. However, due to the limited glucose supply and high dependence on anaerobic glycolysis of cells in the low glucose group, they couldn't make effective adaptive compensation. Therefore, cells in the low-glucose group were more vulnerable to the toxicity of metformin. In conclusion, the enhanced inhibitory effect of metformin on PANC-1 cells cultured in low glucose may be due to the up-regulation of the expression of miR-210-5p, then inhibiting anaerobic glycolytic flux and inducing energy stress via repressing the expression of p-PFKFB2 and activity of LDH. ABBREVIATIONS: PC: pancreatic cancer; DM: diabetes mellitus; PFKFB2: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase2; PFK1: phosphofructokinases; LDH: lactate dehydrogenase; F-2,6-BP: fructose 2,6-bisphosphate.

PFKFB2 regulates glycolysis and proliferation in pancreatic cancer cells.

Tumor cells increase glucose metabolism through glycolysis and pentose phosphate pathways to meet the bioenergetic and biosynthetic demands of rapid cell proliferation. The family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) are key regulators of glucose metabolism via their synthesis of fructose-2,6-bisphosphate (F2,6BP), a potent activator of glycolysis. Previous studies have reported the co-expression of PFKFB isozymes, as well as the mRNA splice variants of particular PFKFB isozymes, suggesting non-redundant functions. Majority of the evidence demonstrating a requirement for PFKFB activity in increased glycolysis and oncogenic properties in tumor cells comes from studies on PFKFB3 and PFKFB4 isozymes. In this study, we show that the PFKFB2 isozyme is expressed in tumor cell lines of various origin, overexpressed and localizes to the nucleus in pancreatic adenocarcinoma, relative to normal pancreatic tissue. We then demonstrate the differential intracellular localization of two PFKFB2 mRNA splice variants and that, when ectopically expressed, cytoplasmically localized mRNA splice variant causes a greater increase in F2,6BP which coincides with an increased glucose uptake, as compared with the mRNA splice variant localizing to the nucleus. We then show that PFKFB2 expression is required for steady-state F2,6BP levels, glycolytic activity, and proliferation of pancreatic adenocarcinoma cells. In conclusion, this study may provide a rationale for detailed investigation of PFKFB2's requirement for the glycolytic and oncogenic phenotype of pancreatic adenocarcinoma cells.

PP2Acα inhibits PFKFB2-induced glycolysis to promote termination of liver regeneration.

The mechanisms underlying the initiation and proliferation of liver regeneration (LR) has been extensively studied using the partial hepatectomy (PHx) model, while little is known about the termination of LR. PP2Acα (protein phosphatase 2 A catalytic subunit α isoform) is the catalytic subunit of protein phosphatase 2 A (PP2A), accounting for most of intracellular serine/threonine phosphatase activity. We have previously observed that termination of LR delayed in PP2Acα liver-specific knockout (LKO) mice after PHx. In our study, we used phospho explorer antibody array analysis to screen the potential phosphorylation targets of PP2Acα, and PP2Acα had a great influence on the hepatic phosphoproteomic signaling in the termination of LR after PHx. We then tested the phosphorylation changes and metabolic function of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-2 (PFKFB2), an isoform of the key glycolytic enzyme PFKFB, which was significantly regulated by PP2Acα knockout. PP2Acα knockout enhanced glycolysis in vivo and in vitro, while adenoviral-mediated RNAi of PFKFB2 reversed the extension of postoperative liver regeneration in KO mice along with the downregulation of glycolysis. Therefore, we demonstrated that PP2Acα liver-specific knockout regulated the hepatocytes glycolysis via activating PFKFB2, thus enhancing liver regeneration during the termination stage.

miR-613 inhibits Warburg effect in gastric cancer by targeting PFKFB2.

miR-613 has been demonstrated to play critical roles in tumorigenesis and progression of a various type of cancers. However, its role and expression significance remain unclear in gastric cancer (GC). We detected the expression of miR-613 in 176 paired GC tissues and adjacent normal tissues, and found that miR-613 was significantly downregulated in GC tissues and its downregulation was correlated with T stage, lymph node invasion and advanced AJCC stages. Moreover, miR-613 expression could be an independent prognostic factor of GC. Biological function analysis indicated that miR-613 inhibited cell proliferation and invasion. Further analysis suggested that miR-613 inhibited Warburg effect of GC cells. Mechanically, we identified that miR-613 could directly bind to the 3'UTR of PFKFB2, thereby suppressing the expression of PFKFB2, which in turn, regulating glycolysis metabolism and cell growth. In conclusion, miR-613 served as a tumor suppressor by targeting PFKFB2, indicating that detecting miR-613 and modulation of miR-613 expression could be potential marker and clinical approach in GC patients.

PFKFB2 Promoter Hypomethylation as Recurrence Predictive Marker in Well-Differentiated Thyroid Carcinomas.

Despite the low mortality rates, well-differentiated thyroid carcinomas (WDTC) frequently relapse. BRAF and TERT mutations have been extensively related to prognosis in thyroid cancer. In this study, the methylation levels of selected CpGs (5-cytosine-phosphate-guanine-3) comprising a classifier, previously reported by our group, were assessed in combination with BRAF and TERT mutations. We evaluated 121 WDTC, three poorly-differentiated/anaplastic thyroid carcinomas (PDTC/ATC), 22 benign thyroid lesions (BTL), and 13 non-neoplastic thyroid (NT) tissues. BRAF (V600E) and TERT promoter (C228T and C250T) mutations were tested by pyrosequencing and Sanger sequencing, respectively. Three CpGs mapped in PFKFB2, ATP6V0C, and CXXC5 were evaluated by bisulfite pyrosequencing. ATP6V0C hypermethylation and PFKFB2 hypomethylation were detected in poor-prognosis (PDTC/ATC and relapsed WDTC) compared with good-prognosis (no relapsed WDTC) and non-malignant cases (NT/BTL). CXXC5 was hypomethylated in both poor and good-prognosis cases. Shorter disease-free survival was observed in WDTC patients presenting lower PFKFB2 methylation levels (p = 0.004). No association was observed on comparing BRAF (60.7%) and TERT (3.4%) mutations and prognosis. Lower PFKFB2 methylation levels was an independent factor of high relapse risk (Hazard Ratio = 3.2; CI95% = 1.1⁻9.5). PFKFB2 promoter methylation analysis has potential applicability to better stratify WDTC patients according to the recurrence risk, independently of BRAF and TERT mutations.

Long non-coding RNA UCA1/miR-182/PFKFB2 axis modulates glioblastoma-associated stromal cells-mediated glycolysis and invasion of glioma cells.

Glioblastoma (GB) is the most common and deadliest malignant primary brain tumor with a high recurrence. In this study, lncRNA UCA1/miR-182 axis has been regarded as a nodal driver of glioma invasion mediated by GB-associated stromal cells (GASCs) and GASC-secreted chemokine CXCL14. In clinical specimens, CXCL14 upregulation in GASCs also correlated with poor prognosis. Notably, CXCL14-high GASCs mediated lncRNA UCA1 upregulation and miR-182 downregulation in glioma cells. Moreover, miR-182 directly bound to the fructose-2,6-biphosphatase PFKFB2; UCA1/miR-182 axis thereby modulated GASC-induced glycolysis in glioma cells. Overall, UCA1/miR-182/PFKFB2 axis modulates chemokine CXCL14 secretion, glycolysis and invasion of glioma cells in GASCs.

UCP2 overexpression enhanced glycolysis via activation of PFKFB2 during skin cell transformation.

Uncoupling protein 2 (UCP2) is an inner mitochondrial membrane transporter which is often upregulated in human cancers. However, how this anion transporter affects tumorigenesis is not well understood. Using the skin cell transformation JB6 model, we demonstrated that UCP2 overexpression activated phosphofructokinase 2/fructose-2,6-bisphosphatase 2 (PFKFB2), a key regulator of glycolysis. In conjunction, upregulation of PFKFB2 expression correlated with elevated fructose 2,6-bisphosphate (Fru-2,6-P2) levels, 6-phosphofructo-1-kinase (PFK-1) activity, glucose uptake, and lactate production. Inhibiting PFKFB2 expression suppressed UCP2-mediated skin cell transformation, decreased cell proliferation, and enhanced mitochondrial respiration, while dampening aerobic glycolysis. The AKT signaling pathway was activated in the UCP2 overexpressed cells; furthermore, the activated AKT signaling contributed to the activation of PFKFB2. Whereas AKT inactivation blocked PFKFB2 activation, suggesting that AKT activation is an important step in PFKFB2 activation. Collectively, our data suggest that UCP2 is a critical regulator of cellular metabolism during cell transformation. Our data also demonstrate a potentially novel mechanism to understand UCP2's tumor-promoting role, which is through the AKT-dependent activation of PFKFB2 and thereby, the metabolic shift to glycolysis (the Warburg effect).

Crystal structure of heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB2) and the inhibitory influence of citrate on substrate binding.

The heart-specific isoform of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB2) is an important regulator of glycolytic flux in cardiac cells. Here, we present the crystal structures of two PFKFB2 orthologues, human and bovine, at resolutions of 2.0 and 1.8 Å, respectively. Citrate, a TCA cycle intermediate and well-known inhibitor of PFKFB2, co-crystallized in the 2-kinase domains of both orthologues, occupying the fructose-6-phosphate binding-site and extending into the γ-phosphate binding pocket of ATP. This steric and electrostatic occlusion of the γ-phosphate site by citrate proved highly consequential to the binding of co-complexed ATP analogues. The bovine structure, which co-crystallized with ADP, closely resembled the overall structure of other PFKFB isoforms, with ADP mimicking the catalytic binding mode of ATP. The human structure, on the other hand, co-complexed with AMPPNP, which, unlike ADP, contains a γ-phosphate. The presence of this γ-phosphate made adoption of the catalytic ATP binding mode impossible for AMPPNP, forcing the analogue to bind atypically with concomitant conformational changes to the ATP binding-pocket. Inhibition kinetics were used to validate the structural observations, confirming citrate's inhibition mechanism as competitive for F6P and noncompetitive for ATP. Together, these structural and kinetic data establish a molecular basis for citrate's negative feed-back loop of the glycolytic pathway via PFKFB2. Proteins 2016; 85:117-124. © 2016 Wiley Periodicals, Inc.