How Can The Synthesis And Breakdown Of Fructose-2,6-bisphosphate Be Controlled Independently?
Oncotarget. 2014 Aug; 5(16): 6670–6686.
Fructose-2,6-Bisphosphate synthesis by 6-Phosphofructo-2-Kinase/Fructose-2,vi-Bisphosphatase four (PFKFB4) is required for the glycolytic response to hypoxia and tumor growth
Jason Chesney
i James Graham Brown Cancer Centre, Departments of Medicine (Hematology/Oncology), Pediatrics and Biochemistry and Molecular Biology, University of Louisville, Louisville, KY
Jennifer Clark
1 James Graham Brown Cancer Center, Departments of Medicine (Hematology/Oncology), Pediatrics and Biochemistry and Molecular Biology, University of Louisville, Louisville, KY
Alden C. Klarer
ane James Graham Dark-brown Cancer Center, Departments of Medicine (Hematology/Oncology), Pediatrics and Biochemistry and Molecular Biology, University of Louisville, Louisville, KY
Yoannis Imbert-Fernandez
1 James Graham Dark-brown Cancer Eye, Departments of Medicine (Hematology/Oncology), Pediatrics and Biochemistry and Molecular Biological science, Academy of Louisville, Louisville, KY
Andrew N. Lane
2 Electric current address: Markey Cancer Center, Academy of Kentucky, Lexington, KY
Sucheta Telang
1 James Graham Brown Cancer Heart, Departments of Medicine (Hematology/Oncology), Pediatrics and Biochemistry and Molecular Biology, University of Louisville, Louisville, KY
Received 2014 Jul 3; Accustomed 2014 Jul 11.
Abstract
Fructose-2,half dozen-bisphosphate (F2,6BP) is a shunt product of glycolysis that allosterically activates half-dozen-phosphofructo-1-kinase (PFK-1) resulting in increased glucose uptake and glycolytic flux to lactate. The F2,6BP concentration is dictated past four bifunctional 6-phosphofructo-2-kinase/fructose-2,vi-bisphosphatases (PFKFB1-4) with distinct kinase:phosphatase activities. PFKFB4 is over-expressed in human cancers, induced by hypoxia and required for survival and growth of several cancer cell lines. Although PFKFB4 appears to be a rational target for anti-neoplastic drug development, information technology is non clear whether its kinase or phosphatase activity is required for cancer cell survival. In this study, we demonstrate that recombinant human being PFKFB4 kinase activity is 4.iii-fold greater than its phosphatase activity, siRNA and genomic deletion of PFKFB4 decrease F2,6BP, PFKFB4 over-expression increases F2,6BP and selective PFKFB4 inhibition in vivo markedly reduces F2,6BP, glucose uptake and ATP. Last, we find that PFKFB4 is required for cancer cell survival during the metabolic response to hypoxia, presumably to enable glycolytic production of ATP when the electron send chain is not fully operational. Taken together, our data signal that the PFKFB4 expressed in multiple transformed cells and tumors functions to synthesize F2,6BP. We predict that pharmacological disruption of the PFKFB4 kinase domain may have clinical utility for the handling of homo cancers.
Keywords: Glycolysis, 6-Phosphofructo-ii-Kinase, Fructose-ii,6-Bisphosphatase, Prostate Cancer, Lung Cancer
INTRODUCTION
Glucose metabolism is regulated past a family unit of four bifunctional half dozen-phosphofructo-2-kinase/ fructose-2,half-dozen-bisphosphatases (PFKFB1-4) that determine the concentration of fructose ii,6-bisphosphate (F2,6BP), which is a strong allosteric activator of the glycolytic enzyme 6-phosphofructo-one-kinase (PFK-1) [1]. Whereas F2,6BP activates PFK-1, several metabolic products including ATP, H+ ions and citrate, allosterically inhibit PFK-ane, indicating that PFK-1 is an essential metabolic sensor in the glycolytic pathway [two]. Given the key role that the PFKFB family members play in regulating glucose metabolism via PFK-1, understanding their specific functions within cells and tissues is of paramount importance to the study of a multitude of affliction states caused by inflammation, ischemia and neoplastic transformation.
Within v years of the initial discovery of F2,6BP in 1980 [3, 4], multiple PFKFB enzyme activities were detected in several organs that were distinguished by having markedly reduced but withal detectable fructose-two,6-bisphosphatase activities relative to the liver PFKFB1 family unit member which has a near 1:ane kinase:bisphosphatase ratio (1). Interestingly, a distinct PFKFB action was detected in the testes [5] and a man testes-specific PFKFB4 gene and then was cloned in 1999 [six] suggesting that the unique metabolic requirements of sperm may necessitate a divergent PFKFB family member. Although several reports indicated that the testes PFKFB4 from diverse species display approximately 3-v fold college kinase than phosphatase action [v, 7, 8], suggesting that its office was predominantly to increase glycolytic flux into the 3-carbon portion of the pathway, i study of purified protein reported a about 1:ane ratio like to that of the liver PFKFB1 family unit member [9].
In the early on 2000s, Minchenko et al. demonstrated that the testes PFKFB4 was induced by hypoxia in multiple cancer cell lines and over-expressed in matched human lung, chest and colon tumor tissues relative to normal tissues from the same patients [10-13]. The functional requirement of PFKFB4 for tumor growth then was published in 2010 when researchers demonstrated that selective inhibition of PFKFB4 with siRNA suppressed the growth of human lung adenocarcinoma xenografts in athymic mice (U.South. Patent #eight,283,332; PFKFB4 Inhibitors and Methods of Using the Same). The importance of PFKFB4 every bit a potential target for the development of cancer therapeutics was then significantly expanded in 2012 by two contained groups that conducted unbiased screens for genes essential for cancer survival and found that PFKFB4 is required for both glioma stem-like jail cell [14] and prostate cancer cell survival [15] but non for normal cell survival. Taken together, these studies indicated that PFKFB4 may be a useful molecular target for the evolution of anti-neoplastic agents.
An essential start step in developing anti-cancer agents that inhibit PFKFB4 is to decide whether the kinase or phosphatase domain of this bifunctional enzyme are active in neoplastic cells. Whereas the kinase activity will produce F2,6BP and increase flux downwards the glycolytic pathway producing ATP, the phosphatase activeness will decrease F2,6BP, reduce PFK-i activeness and increase the oxidative pentose shunt action and NADPH via the availability of glucose 6-phosphate which is in equilibrium with fructose 6-phosphate (F6P) through glucose half-dozen-phosphate isomerase. Given that the intracellular concentration of the substrate of the kinase domain (F6P) is >10,000 fold the substrate of the phosphatase domain (F2,6BP) in transformed cells (e.g. MCF-7 cells: F6P, fifty±24 nmol/mg poly peptide [xvi]; F2,6BP, two.97±.21 pmol/mg protein [17]), we predicted that the kinase activity of PFKFB4 would be the dominant office of this enzyme in transformed cells.
In the present written report, nosotros find that PFKFB4 functions primarily to produce F2,6BP, thus stimulating glycolytic flux to lactate and the Kreb's cycle in order to generate ATP and anabolic precursors. Appropriately, the synthesis of small molecule inhibitors of the PFKFB4 kinase domain is anticipated to enable the development of novel agents for the treatment of cancer.
RESULTS
High Expression of PFKFB4 in Several Normal Tissues and in Lung Adenocarcinomas
Nosotros first conducted a simultaneous analysis of PFKFB1-4 mRNA expression in normal man tissues using real-time RT-PCR for each family unit member. Nosotros observed high expression of: PFKFB1 in liver and skeletal musculus; PFKFB2 in eye, lung, skeletal muscle, kidney, pancreas and testes; and PFKFB4 in placenta, lung, skeletal muscle, pancreas, spleen, prostate, testes, ovary, colon and leukocytes (Fig. 1A). PFKFB3, which has been characterized every bit being inducible [xviii] and ubiquitous [xix], was expressed at very low levels in all tissues with the exception of leukocytes (Fig 1A). We so used multiplex RT-PCR and real-time RT-PCR to appraise PFKFB1-4 mRNA expression in normal lung tissues and adjacent lung adenocarcinomas from v patients. Only PFKFB4 mRNA was over-expressed in each of these patients' tumors (Fig. 1B, C). Farther test of a larger cohort of lung adenocarcinomas and adjacent normal tissues from 18 patients by immunohistochemical analyses revealed high PFKFB4 protein expression relative to normal lung alveoli (Fig. 1D, East).
PFKFB4 Is Expressed in Multiple Normal Tissues and Over-Expressed in Human Lung Adenocarcinomas
PFKFB1-4 mRNA expression in multiple normal tissues was assessed by real-time RT-PCR and expressed by copy number (A). Matched normal (N) and lung adenocarcinoma (T) tissue were analyzed past multiplex RT-PCR (B) and by existent-time RT-PCR expressed as fold change relative to adjacent normal tissues (C). Immunohistochemical staining of lung adenocarcinomas and normal lung was conducted on patients' biopsies, three representative sections are shown (D) and positive pixels were quantitated (E). Data are expressed as the hateful ± SD of three experiments. *p value < 0.001 compared to normal tissue controls.
The Kinase:Phosphatase Ratio of PFKFB4 is 4.three:1 and Selective Inhibition of PFKFB4 Expression Reduces F2,6BP in Multiple Cancer Cell Lines
We expressed and purified recombinant human being PFKFB4 and compared the kinase and phosphatase activities to those of the inducible PFKFB3 recombinant poly peptide. Given the recent reports that PFKFB4 functions as a dominant bisphosphatase in cancer cells [xv], we were surprised to detect that recombinant human being PFKFB4 had significant kinase action that was ~xv% that of the PFKFB3 family fellow member whose kinase domain has previously been established to prepare the intracellular concentration of F2,6BP in multiple prison cell lines (PFKFB4 kinase Vmax = 5.06±0.2 mU/mg, PFKFB3 kinase 5max= 38.4 ±2.3 mU/mg) (Fig. 2A) [18, 20, 21]. In contrast, both PFKFB4 and PFKFB3 were found to have relatively low phosphatase activities (PFKFB4 bisphosphatase Fivemax = 1.17±0.02 mU/mg; PFKFB3 bisphosphatase Fivemax = 0.47±0.035 mU/mg) and the kinase:bisphosphatase ratios for PFKFB4 and PFKFB3 were calculated to be 4.3:1 and 81.7:ane, respectively (Fig. 2A). Since the concentration of the kinase substrate, F6P, is so much higher than that of the bisphosphatase substrate, F2,6BP (e.m. H460 cell [F6P] = 12518±1104 pmol/mg poly peptide; [F2,6BP] = 4.47±.21 pmol/mg protein) and because the kinase:phosphatase is 4.three:1, we predicted that selective inhibition of PFKFB4 would preferentially inhibit the kinase function of this bifunctional enzyme and thus result in a reduction in F2,6BP. We found that this did indeed occur in half-dozen out of vii transformed cancer prison cell lines derived from lung, colon, prostate and breast that were each transfected with two separate PFKFB4-specific siRNAs (Fig. 2B). Importantly, genomic deletion of Pfkfb4 in LT-immortalized fibroblasts as well acquired a marked reduction in F2,6BP (Fig. 2C). Since PFKFB3, dissimilar PFKFB4, has been established to function as a kinase in transformed cells, we next examined the event of PFKFB3 siRNA transfection on three transformed prison cell lines and similarly observed a marked reduction in the steady-state concentration of F2,6BP (Fig. 2nd).
Recombinant PFKFB3 and PFKFB4 Kinase and Phosphatase Activities and Effects of PFKFB3 and PFKFB4 Inhibition on Intracellular F2,6BP in Cancer Cells
Human being PFKFB3 and PFKFB4 were expressed and relative kinase and phosphatase specific activities (S.A.) were determined (A). The indicated cancer cell lines were transfected with control siRNA (Ctrl) or two PFKFB4 siRNAs (FB41 and FB42) and, after 48 hours, protein expression examined past Western blot and quantitated by densitometry and intracellular F2,6BP was measured (B). Large T antigen-immortalized, tamoxifen (4OHT)-inducible Pfkfb4 −/− lung fibroblasts were exposed to i nM and 10 μM 4OHT (ethanol used as vehicle control) and analyzed for protein expression and F2,6BP later on 48 hours (C). The indicated cancer prison cell lines were transfected with an established PFKFB3 siRNA and analyzed for protein expression and F2,6BP 48 hours later (D). Data are expressed as the mean ± SD of three experiments. * p value <0.001 compared to vehicle or control siRNA.
Selective Inhibition of PFKFB4 Expression Reduces Glycolysis and ATP in Multiple Cancer Cell Lines
Nosotros examined the issue of the two PFKFB4 siRNA molecules and genomic deletion of Pfkfb4 on glycolysis (measured by the production of iiiH2O from [5-3H]glucose), ATP and NADPH. We predicted that if the kinase function of PFKFB4 is dominant, so PFKFB4 inhibition would suppress (rather than increase) glycolysis and decrease the concentration of ATP which is produced downstream of PFK-1. Nosotros found that both glycolysis and ATP were reduced in all five jail cell lines examined although the ATP depletion in MCF-seven cells was not statistically meaning (Fig. 3A). Although we observed variable effects on NADPH, nosotros did find that PFKFB4 inhibition increased NADPH in H460, A549, LNCaP and MCF-7 cells, presumably equally a result of increased availability of F6P for the activity of glucose 6-phosphate isomerase and the oxidative pentose shunt (Fig. 3A). Importantly, we plant that Pfkfb4 genomic deletion reduced glycolysis and ATP and increased NADPH, all findings consistent with a requirement of PFKFB4 for the activation of PFK-ane (Fig. 3B).
Effects of PFKFB4 Inhibition on Cancer Prison cell Glycolysis, ATP and NADPH
The indicated cell lines were transfected with control siRNA (Ctrl) or two PFKFB4 siRNAs (FB4i and FB4two) and glycolysis (3H2O product from [v-3H]glucose), intracellular ATP and NADPH were measured (A). Large T antigen-immortalized, tamoxifen (4OHT)-inducible Pfkfb4 −/− lung fibroblasts were exposed to ane nM and x μM 4OHT and analyzed for glycolysis, ATP and NADPH 48 hours later (B). Data are expressed every bit the hateful ± SD of three experiments. * p value <0.01 compared to vehicle or control siRNA.
Over-Expression of PFKFB4 Increases F2,6BP, Glycolysis and ATP and reduces NADPH
Given that the kinase activity of PFKFB4 is four.3-fold the phosphatase action and that PFKFB4 siRNA transfection and genomic deletion both reduced F2,6BP, we predicted that over-expression would accept the opposite effect on F2,6BP and subsequent metabolic events. We plant that PFKFB4 over-expression increased F2,6BP, glycolysis and ATP, and decreased NADPH in each of the examined transformed cell lines (Fig. iv). We also examined the effect of addition of 5 mM pyruvate on the depletion of ATP caused by PFKFB4 siRNA in H460 cells and found that pyruvate partially reversed the ATP depletion, further supporting a office for PFKFB4 in increasing the glycolytic product of ATP (ATP, pmoles/μg protein: Ctrl siRNA, 39.9±0.ii; PFKFB4 siRNA ix.94±0.5; Ctrl siRNA + Pyruvate, 40.4±0.13; PFKFB4 siRNA + pyruvate, 33.1± 1.i; p value < 0.01).
Over-Expression of PFKFB4 in Cancer Cells Increases F2,6BP, Glycolysis and ATP but Decreases NADPH
The indicated cancer cell lines were transfected with either empty pCMV-XL4 (Vec) or pCMV-XL4 containing full-length PFKFB4 (+FB4) and analyzed 48 hours afterward past Western blot and densitometry, and for F2,6BP, glycolysis, ATP and NADPH. Data are expressed every bit the mean ± SD of iii experiments. * p value < 0.01 compared to empty pCMV-XL4 vector.
Inhibition of PFKFB4 Suppresses H460 Lung Adenocarcinoma F2,6BP, Glucose Uptake and Growth In Vivo
In order to decide if PFKFB4 functions to synthesize F2,6BP in vivo, we stably transfected H460 cells with PFKFB4 shRNA and confirmed reduced PFKFB4 poly peptide expression in vitro (Fig 5A). We found that stable PFKFB4 inhibition reduced anchorage-independent growth as soft agar colonies and as tumors (Fig. 5A, B). We then examined resected tumors afterwards two weeks of growth and observed a marked reduction in tumor F2,6BP and ATP and an increase in apoptotic cells in the tumors by immunohistochemistry for cleaved caspase 3 (Fig 5C, D, F) We also establish a pocket-size increase in NADPH in the PFKFB4 shRNA tumors in comparison with control tumors which was non statistically significant (Fig 5E). In addition, we examined uptake of [eighteenF]-FDG in PFKFB4 shRNA and control tumors by PET browse and observed reduced tumor [eighteenF]-FDG uptake in the PFKFB4 shRNA tumors (Fig. 5G) as has also been described as a event of PFKFB3 inhibition. Taken together, these in vivo studies provide stiff evidence that PFKFB4 supports tumor growth by functioning equally a kinase to synthesize F2,6BP.
Stable shRNA Knock-Down of PFKFB4 Reduces Tumor Growth, Glucose Uptake and F2,6BP, and Increases Apoptosis
H460 cells were stably transduced with PFKFB4 (shFB4) or control (Control, Ctrl) shRNA and assessed for PFKFB4 and PFKFB3 protein expression by Western blot analysis (A), soft agar colony germination (A), tumor growth in athymic mice (B). F2,6BP concentration (C), ATP (D), and NADPH (E) were measured in tumors post-obit resection. Data are expressed as the hateful ± SD of three experiments. Tumor sections were examined for apoptosis past cleaved caspase iii (CC3) immunohistochemistry, with representative images on the left and positive pixels quantified on the right (F). The data is depicted as % positive pixels/full pixels ± SD. Tumors in mice were examined for 18F-FDG uptake in vivo past micro-PET imaging. Regions of interest in the tumors and cerebellum were quantified in quadruplicate (right) and representative transverse cuts are shown on the left (Thousand). *p value < 0.001 compared to control shRNA.
PFKFB4 mRNA and Protein Expression Are Increased past Hypoxia and Required for Hypoxia-Induced F2,6BP, Glucose Uptake and Glycolysis
Both PFKFB3 and PFKFB4 mRNA previously have been found to be induced past hypoxia [x, 12, 13, 22-24] and we confirmed these by observations in H460 cells (Fig. 6A). In order to compare the functional relevance of PFKFB3 and PFKFB4 under hypoxic conditions, nosotros transfected H460 cells with PFKFB4 or PFKFB3 siRNA under 21% and ane% oxygen. Nosotros observed a reduction in the hypoxic induction of both enzyme mRNAs every bit a effect of the siRNA molecules (Fig. 6A). Hypoxia strongly induced PFKFB4 protein expression, as has been observed in other jail cell lines (Fig. 6A-C). Interestingly, we found a far lower induction of PFKFB3 protein which may already be maximally expressed equally a result of the multitude of genetic alterations that increase PFKFB3 protein expression such as PTEN loss and Ras activation (east.m. H460 cells express KRASQ61H ) [20, 25, 26]. Chiefly, transfection with either PFKFB4 or PFKFB3 siRNA acquired an attenuation of F2,6BP, glucose uptake and glycolysis stimulated by hypoxia (Fig. 6D-F). The concentrations of both ATP and NADPH were decreased under hypoxia and PFKFB4 siRNA exacerbated the ATP decrease, indicating that PFKFB4 may be essential for the glycolytic response to hypoxia in these cells (Fig. 6G, H).
Role of PFKFB3 and PFKFB4 in Hypoxia-Induced F2,6BP production, Glucose Uptake, Glycolysis, ATP and NADPH
H460 cells were transfected with control siRNA (siCtrl), PFKFB3 siRNA (siFB3) or PFKFB4 siRNA (siFB4), cultured in 21% oxygen or 1% oxygen and examined for PFKFB3 and PFKFB4 mRNA expression (A) and protein expression by Western absorb analysis and densitometry (B and C), F2,6BP (D), 14C-ii-DG uptake (E), glycolysis (threeH2O production from [5-iiiH]glucose) (F), intracellular ATP (M) and NADPH (H). Data are expressed as the mean ± SD of iii experiments. * p value < 0.01 compared to control siRNA.
PFKFB3 or PFKFB4 Inhibition in H460 Cells Reduces Glycolytic Flux to Lactate and Glutamate
Given that both PFKFB3 and PFKFB4 siRNA suppressed hypoxia-induced F2,6BP, nosotros suspected that inhibition of PFKFB3 or PFKFB4 would outcome in reduced glycolytic flux to lactate and into the TCA cycle product, glutamate, under hypoxic atmospheric condition. Whereas we found that both PFKFB3 and PFKFB4 siRNAs caused a reduction in the conversion of 13C-glucose to lactate and glutamate equally assessed by NMR, the PFKFB4 siRNA caused a far greater reduction in the conversion to both products under both normoxic and hypoxic conditions (Fig. 7A, B).
Role of PFKFB3 and PFKFB4 in the Conversion of xiiiC-Glucose into 13C-Lactate and 13C-4-Glutamate
H460 cells were transfected with control siRNA (siCtrl), PFKFB3 siRNA (siFB3) or PFKFB4 siRNA (siFB4), cultured in 21% oxygen or 1% oxygen, pulsed with uniformly-labeled 13C-glucose for hours and extracted for NMR analyses of xiiiC-lactate and 13C-four-glutamate. (A) 1D NMR spectra of media extracts showing the region corresponding to the methyl groups of lactate and essential amino acids. Satellite peaks were integrated and normalized to internal DSS concentration to give the concentrations noted. Nether these conditions, nearly all of the lactate derives from glucose. (B) 1D aneH{xiiiC} HSQC spectra of cellular extracts showing selective enrichment at the C4 cantlet of glutamate (Glu) under normoxia, (21 % O2, left) and under hypoxia (1% Otwo, right). The C4 position of Glu becomes enriched past addition of glucose-derived acetate to oxaloacetate. The resulting 13C2 citrate is converted by the TCA bike to 2-oxoglutarate and transaminated to Glu, showing that the TCA cycle remains active in these cells, but is profoundly reduced under one% oxygen and by PFKFB3 and PFKFB4 knockdown. Values on the right denote ratios of pinnacle areas normalized to uridine ribose (Urib) H1', which correspond to relative amounts of glucose-derived 13C. Numbers expressed are the mean ± SD of three experiments.
PFKFB3 or PFKFB4 Inhibition in H460 Cells Increases Apoptosis Under Normoxic and Hypoxic Conditions
Maintenance of intracellular ATP is required to prevent apoptosis and we postulated that PFKFB3 or PFKFB4 inhibition would increase apoptosis. Nosotros observed a marked increase in apoptotic PI+/Annexin V+ cells and cleaved PARP with inhibition of either family unit member nether both normoxia and hypoxia but observed the greatest number of apoptotic cells as a outcome of the combination of hypoxia and PFKFB4 inhibition (Fig. 8A-C).
Issue of PFKFB3 or PFKFB4 siRNA Transfection on Apoptosis
H460 cells were transfected with control siRNA (siCtrl), PFKFB3 siRNA (siFB3) or PFKFB4 siRNA (siFB4), cultured in 21% oxygen or i% oxygen for 48 hours, and analysed for apoptosis by period cytometry of propidium iodide and annexin Five positive cells (A and B, PI+ + PI/Ann V+ cells shown as % apoptotic cells) and by cleaved PARP by Western blot assay and densitometry (C). Data are expressed every bit the mean ± SD of three experiments. * p value < 0.005 compared to control siRNA.
PFKFB4 Expression Correlates With Hypoxia in Man Lung Adenocarcinoma Xenografts
We examined serial sections of human H460 lung adenocarcinoma xenograft tumors resected from athymic mice for expression of PFKFB4 and PFKFB3 and correlated the expression levels of these proteins using carbonic anhydrase 9, a potent transcriptional target of HIF-1α, as a hypoxia mark. Nosotros observed a statistically pregnant correlation between PFKFB4 and carbonic anhydrase Ix expression but observed no correlation betwixt PFKFB3 and carbonic anhydrase Ix (Effigy 9A-D).
PFKFB4 Expression Correlates with Hypoxia In Human Lung Adenocarcinoma Xenografts
Ten human being H460 lung adenocarcinoma xenografts were analyzed past immunohistochemistry for carbonic anhydrase (CA) IX, PFKFB3 and PFKFB4. Representative adjacent sections from two tumors are provided (A,B). 20X magnification of portion of tumor in (B) shown (C). Positive pixels were enumerated in a minimum of 5 fields per tumor section followed by linear regression analysis (D). r2 and p values are provided.
DISCUSSION
The primary objective of this study was to determine which of the two enzymatic domains of PFKFB4 is active in transformed cells. Using multiple jail cell types and approaches, we plant that: (i) recombinant homo PFKFB4 has far more than kinase activity than phosphatase activity (kinase:phosphatase ratio = 4.3:1); (ii) selective inhibition of PFKFB4 using ii unique siRNA molecules reduces the intracellular F2,6BP in six of seven cancer cell lines indicating that endogenous PFKFB4 synthesizes F2,6BP in these cancer cells; (iii) over-expression of PFKFB4 increases the F2,6BP in all examined cancer cell lines; and (four) genomic deletion of the Pfkfb4 cistron reduces F2,6BP in LT-immortalized fibroblasts. Taken together, these in vitro data provide overwhelming bear witness that the main biochemical function of PFKFB4 is to synthesize F2,6BP. We also establish that PFKFB4 siRNA reduces glycolysis and ATP and increases the steady-state concentration of NADPH in vitro, all expected metabolic effects of reduced F2,6BP. Additionally, we observed that PFKFB4 shRNA suppressed the glucose uptake, F2,6BP, ATP and growth of H460 xenograft tumors in mice, thus providing further testify that the main function of PFKFB4 in vivo is to synthesize F2,6BP and thus actuate PFK-1 and glycolysis. The rescue of transformed cells from PFKFB4 inhibition with the addition of pyruvate, which provides substrate for mitochondrial ATP production, provides further back up that PFKFB4 is predominantly functioning to stimulate ATP product. To summarize, these data indicate that the kinase activity and, thus, the capacity to produce F2,6BP, serves as the dominant office of PFKFB4 in cancer cells.
Since the majority of human cancers display elevated glucose uptake and several targeted agents such equally the BRAF inhibitor, vemurafenib, and the estrogen antagonist, fulvestrant, acutely suppress glucose metabolism in club to inhibit tumor growth [17, 27], we believe that PFKFB3 and PFKFB4 kinase inhibitors may prove effective for the treatment of cancer. Importantly, a novel family of related small molecule antagonists of the kinase domain of PFKFB3 (i.e. 3PO, PFK15 and PFK158) have been found to reduce glycolytic flux, ATP and cancer prison cell viability [17, 21, 28]. PFK158 is at present beingness tested in a phase I clinical trial of advanced cancer patients (clinicaltrials.gov #{"blazon":"clinical-trial","attrs":{"text":"NCT02044861","term_id":"NCT02044861"}}NCT02044861) and recently has been found to synergistically interact with targeted cancer agents [17]. Given that PFKFB3 and PFKFB4 are co-expressed in several cancer cell lines and have the redundant function of synthesizing F2,6BP, we postulate that selective inhibition of a single family unit member may result in bounty past a co-expressed PFKFB family fellow member, and that combinations of PFKFB3 and PFKFB4 inhibitors may be necessary in order to fully suppress tumor glucose metabolism and growth. Time to come studies will be directed at creating double Pfkfb3 and Pfkfb4 knockout mice in club to conduct in vivo studies of the roles of these 2 seemingly redundant enzymes in regulating the glucose metabolism and growth of tumors.
Although this is the get-go comprehensive assay of the relative importance of the kinase activity of PFKFB4 in transformed cells to include a measurement of homo recombinant protein action and a combination of siRNA knock-down, plasmid-based over-expression and genomic deletion experiments in multiple cancer cell types, two prior studies previously have supported the conclusion that the kinase domain of PFKFB4 was essential for cancer cell proliferation. A PFKFB4 siRNA was found to reduce F2,6BP and glycolytic flux to lactate in A549 lung adenocarcinoma cells (U.S. Patent #8,283,332) and two other PFKFB4 siRNAs were independently observed to reduce lactate secretion and the intracellular ATP in malignant glioma cells [14]. Nonetheless, a 3rd study establish that PFKFB4 siRNA increased the F2,6BP concentration of three prostate cancer prison cell lines [15] including the LNCaP and PC3 cell lines (using one of the ii siRNAs examined in the current study). Although it is difficult to reconcile the findings from this single discordant report with those of the other two studies as well every bit the present study, we believe that the combined utilize of recombinant poly peptide analyses, cell lines derived from several types of cancer, two methods to inhibit PFKFB4 (siRNA and genomic deletion) and PFKFB4 over-expression experiments provide comprehensive and compelling information demonstrating that the kinase activity of PFKFB4 dictates the F2,6BP concentration in transformed cells. Whereas we look forward to connected independent examinations of the biochemical functions of PFKFB4 in transformed cells, our current data back up an essential role of the PFKFB4 kinase activeness for cancer cell survival as well equally the development of small-scale molecule antagonists that target the kinase domain of PFKFB4.
Although the differences in metabolic functions of PFKFB3 and PFKFB4 are poorly understood, we observed a more robust consecration of PFKFB4 poly peptide relative upon exposure of H460 cells to i% oxygen and significant correlation between hypoxia and PFKFB4 expression in lung adenocarcinoma tissues. Additionally, we detected a greater reduction in glycolytic flux to glutamate and lactate caused by PFKFB4 siRNA relative to PFKFB3 siRNA transfection. Given these results, we doubtable that PFKFB4 may serve as a unique regulator of the glycolytic response to hypoxia. This hypothesis is supported by the observation that the H460 cells underwent increased hypoxia-associated apoptosis when subjected to PFKFB4 siRNA transfection but not when subjected to PFKFB3 siRNA transfection. These information suggest that tumors that are especially afflicted by poor vasculature and hypoxia may be about responsive to PFKFB4 inhibition.
CONCLUSIONS
In conclusion, our data indicate that the PFKFB4 family member functions predominantly to synthesize F2,6BP which, in turn, is required for glycolytic flux through PFK-one and subsequent ATP product. This conclusion is consistent with the finding that H460 cells are exquisitely sensitive to PFKFB4 inhibition under hypoxic weather condition when the efficiency of ATP production by the mitochondria is compromised and the reliance on glycolysis to produce ATP is greatest. With the key intramolecular target of PFKFB4 now established, we believe that PFKFB4 inhibitors can be rationally designed and that these agents may prove most useful when combined with PFKFB3 inhibitors in order to circumvent potential bounty betwixt these two PFKFB family members.
METHODS
Jail cell lines and cell culture
A549 and H460 NSCLC, MCF7 breast adenocarcinoma, LNCaP, PC3 and DU145 prostatic adenocarcinoma and HCT116 colon adenocarcinoma cell lines were obtained from ATCC (Manassas, VA). PFKFB4−/− ear pinna fibroblasts were isolated from TamCre/loxP/PFKFB4−/− mice and immortalized by transduction with REBNA/IRES retrovirus expressing SV40 large T antigen as described previously [20]. Cell lines were grown in DMEM (A549, LNCaP, PC3, DU145 and PFKFB4−/−), RPMI 1640 (H460), McCoy's 5A media (HCT116) or improved MEM (MCF7) (all from Invitrogen, Grand Island, NY) containing x% fetal calf serum (FCS, Hyclone, Logan, UT) at 37ºC in 5% CO2. In some experiments, iv-hydroxytamoxifen (4OHT, 4HT, Sigma -Aldrich, St. Louis, MO) was added to PFKFB4−/− fibroblasts at indicated concentrations.
Transfections
For siRNA experiments, cells growing in 6-well plates were transfected with command siRNA (Stealth Negative Control Medium GC Duplex, Invitrogen), PFKFB3 siRNA (FB3; HSS107860, Invitrogen) or PFKFB4 siRNA (FB41, FB4; HSS107863, Invitrogen and FB4ii; siGENOME human PFKFB4 5210 #ii, Dharmacon) using Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA), and harvested 48 hours later transfection. For hypoxia experiments, cells were transfected with siRNA and, afterwards 24 hours, were placed in a hypoxia chamber (Billups-Rothenburg, Del Mar, CA) purged with one% oxygen for 24 hours. For brusk hairpin RNA (shRNA) experiments, sense and antisense DNA oligonucleotides for PFKFB4 were designed with a hairpin against the target sequences 5′-cacttgtatggtcctgt-3′ and v′-ggagagcgaccatcttt-three′ were produced by IDT (Coralville, IA). The oligonucleotides were annealed and ligated into pSUPER.neo vector (OligoEngine, Seattle, WA) following manufacturer'southward instructions. H460 cells were transfected with an shRNA-expressing plasmid targeted against PFKFB4 (shFB4) or a scrambled shRNA expressing plasmid (Control) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and clones selected with G418 (500 μg/ml). For overexpression experiments, cells were transfected with pCMV-XL4 (vector) or pCMV-XL4 containing full-length PFKFB4 (Origene, Rockville, MD) using Lipofectamine 2000 and harvested afterward 48 hours.
PCR analyses
Multiplex mRNA primers were custom synthesized (IDT) against human PFKFB1-4 equally described previously [20]. cDNA from normal man tissues and matched tumor and adjacent normal tissues (Clontech, Mountain View, CA) were analyzed using these primers and standard PCR weather condition. PFKFB1-4 mRNA expression was determined using real-fourth dimension RT-PCR with TaqMan probes for human PFKFB1-4 and β-actin (Practical Biosystems, Foster City, CA) in triplicate in 96-well optical plates (MicroAMP®, Practical Biosystems). Analysis of results and fold differences between samples were adamant using StepOne software (version2.ane) (Practical Biosystems) and calculated from the ΔΔCT values with the formula (ii−ΔΔCT). The data are represented as the mean ± SD from triplicate measurements from iii independent experiments. For calculation of copy number, the molecular weight was determined for the double stranded Dna sequences of the 4 PFKFB isoforms. The OD of the DNA (in gm/μL) was divided by the molecular weight of the production (event in moles/μL) so this number was multiplied by Avogadro's number (6.022 × 1023 molecules/mole). The resulting number of DNA molecules per μL was used to generate a standard curve from which copy numbers were calculated. Statistical significance was assessed by the two-sample t examination (contained variable).
Protein extraction and Western blotting
Cells were harvested, washed X1 in PBS and lysed in 1X lysis buffer (Pierce Biotechnology, Rockford, IL) containing protease inhibitors. Protein samples were resolved on four-20% SDS-PAGE gels (BioRad, Hercules, CA) and transferred to PVDF membranes (BioRad). After blocking in TBS containing 0.1% Tween 20 (TBS-T) and 5% milk, membranes were probed with antibodies to PFKFB3 (Proteintech, Chicago, IL), PFKFB4 (Abcam, Cambridge, MA) or β-actin (Sigma, St. Louis, MO). Secondary antibodies used were HRP-conjugated goat anti-rabbit or anti-mouse (1:5000, Pierce Biotechnology). Scanned images were quantified by densitometric analyses using Image J software (http://rsb.info.nih.gov/ij/). Values obtained were normalized to β-actin and expressed in densitometric units as a percentage of control. The data represented are the mean ± SD from triplicate measurements from three contained experiments. Statistical significance was assessed past the two-sample t test (independent variable).
Kinase and Bisphosphatase assays
Fructose-6-phosphate kinase and fructose-2,6-bisphosphatase activities of human recombinant PFKFB3 and PFKFB4 were assayed using previously described methods wherein one unit of activity was defined as the amount of enzyme that catalyzes the formation of 1 μmol of F2,6BP per min. [vii, 8]. Aliquots of the reaction mixture were removed at intervals, added to 0.1N NaOH and then neutralized to a pH of 7.two and assayed for F2,6BP as described beneath.
F2,6BP measurements
Cells were harvested, washed twice with PBS, lysed in NaOH/Tris acetate past heating at 80°C for 5 min. Lysates were neutralized to pH 7.2 with ice-cold acetic acrid and HEPES. F2,6BP content was measured using a coupled enzyme reaction following the method of Van Schaftingen et al [29]. The F2,6BP concentration was normalized to full cellular protein measured past the bicinchoninic acrid assay (BCA, Thermo Scientific, Rockford, IL). All data are expressed as the mean ± SD of 3 experiments. Statistical significance was assessed by the 2-sample t exam (independent variable).
Glycolysis analysis
Cells growing in 6-well plates were incubated in 500 μl of consummate medium containing 1 μCi of 5-[iiiH] glucose per well for 60 min in 5% CO2 at 37°C. The medium was then collected and centrifuged for 5 min at 8000 rpm to pellet any suspended cells. To separate the 3H2O formed via glycolysis from the 5-[threeH]glucose added to the medium, an evaporation technique in a sealed system was utilized. Briefly, 150 μl aliquots of medium were added to open tubes that were placed upright inside scintillation vials containing 1 ml of H2O. The scintillation vials were sealed, and the 3H2O produced by glycolysis through enolase and released to the medium was allowed to equilibrate with the H2O in the outer vial for 48 h at 37°C. The amounts of 3HiiO that had diffused into the surrounding HtwoO was measured on a Tri-Carb 2910 liquid scintillation analyzer (Perkin Elmer, Boston, MA) and compared with 3HtwoO and 5-[3H]glucose standards. Poly peptide concentration was determined using the BCA assay and counts were normalized to protein concentration. All information are expressed equally the mean ± SD of three experiments. Statistical significance was assessed by the 2-sample t test (independent variable).
2-[1-14C]-Deoxy-D-Glucose Uptake
Cells were placed in glucose-free media for 30 minutes, 2-[one-14C]-deoxy-D-glucose (0.25 μCi/mL; Perkin Elmer) was added for an additional sixty min and the cells then were washed thrice with water ice-cold glucose-gratuitous media. Prison cell lysates were collected in 500 μL of 0.1% SDS, and scintillation counts (counts/min) were measured on 400 μL of the lysate. Counts were normalized to poly peptide concentration measured past the BCA assay and data are represented as mean ± SD from triplicate measurements from three contained experiments. Statistical significance was assessed past the two-sample t test (independent variable).
ATP Measurements
Cell pellets were lysed using Passive Lysis buffer (1X, Molecular Probes, Invitrogen). Lysates were wink frozen in liquid nitrogen and thawed (to 37ºC) once to achieve complete lysis and so centrifuged at 4ºC for 30 seconds to articulate the lysates. Intracellular ATP levels were determined using a bioluminescence assay (Molecular Probes, Eugene, OR) utilizing recombinant firefly luciferase and its substrate, d-luciferin and following manufacturer's instructions. The luminescence was read in a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA) at 560 nm. The ATP values were calculated using an ATP standard curve. The protein concentrations of the lysates were estimated using the BCA assay and ATP was expressed every bit pmol per μg protein. All data are expressed as the mean ± SD of three experiments. Statistical significance was assessed by the two-sample t test (independent variable).
NADPH Measurements
Cells were harvested, washed with water ice common cold PBS and homogenized in NADPH extraction buffer (EnzyFluo, Bioassay Systems, Hayward, CA). NADPH was then measured following manufacturer's instructions (Bioassay Systems) by reduction of the provided probe into a fluorescent product measured at λex/em of 530/585 nm. Values were compared with a standard curve. All data are expressed as the hateful ± SD of three experiments. Statistical significance was assessed by the two-sample t test (independent variable).
Apoptosis Assay
Cells were stained with FITC-labeled annexin-5 and propidium iodide post-obit the manufacturer'south protocol (BD Biosciences, San Diego, CA). Briefly, cells were detached and 100,000 cells per sample were pelleted past centrifugation at 1500 rpm for 5 minutes and done X1 with PBS. The cells were so resuspended in 1X binding buffer, annexin-V/FITC and/or propidium iodide added and were incubated in the nighttime at room temperature for fifteen minutes. 1X binding buffer was added to increase volume and ten,000 events were counted for each sample using the advisable filters for FITC and PI detection (BD FACSCalibur, San Jose, CA). Data was analyzed using FlowJo software (TREE STAR Inc, San Carlos, CA). Results were calculated as the average of triplicate samples ± SD.
NMR Experiments
Cells treated with siRNA were grown in media containing uniformly labeled 13C-glucose (2 gm/L, Cambridge Isotopes Laboratories, Andover, MA) for 24 hours. Media samples were frozen in liquid nitrogen. Cells were counted and equal numbers of cells were pelleted, washed twice with cold PBS to remove adhering medium, and flash frozen in liquid nitrogen. The cell pellets and media samples were extracted with x% ice-cold trichloracetic acid (TCA) followed past lyophilization. The extracts were redissolved in D2O containing 85 μM (media) or 95 μM (cells) DSS (2, 2-dimethyl-2-silapentane-5-sulfonate sodium salt) as both a chemical shift reference and as a concentration standard and loaded into v mm Shigemi tubes (Shigemi, Tokyo, Nihon). Nuclear magnetic resonance (NMR) spectra were recorded at 14.1 T on a Varian Inova spectrometer at 20°C using a ninety° excitation pulse as described previously [xxx]. 1D proton NMR spectra of extracts were recorded with a 2 sec acquisition fourth dimension and a total recycle time of 5 sec, with presaturation of the residual HOD resonance for 3 seconds. For analyzing the cellular extracts and determining the positional enrichment with xiiiC we used 2d experiments (TOCSY and 1H{13C}-HSQC), and quantified the 13C satellite and 12C peaks by volume integration in the TOCSY using established assignment protocols [xxx, 31]. The NMR experiments were carried out in the Structural Biology Plan's NMR Core at the University of Louisville James Graham Brown Cancer Center.
Mouse Studies
H460 cells stably transfected with either PFKFB4 or control shRNA were nerveless from exponential growth stage culture. Cells were washed twice, re-suspended in PBS (5 × ten7 cells/ml) and groups of x BALB/c athymic female mice were injected s.c. with 100 μL (5 × x6 cells) of the cell suspension. The tumors were followed from the fourth dimension of appearance until the finish of the experiment. Tumor masses were determined in a blinded fashion with Vernier calipers according to the following formula: weight (mg) = (width, mm)ii × (length, mm)/two [32]. All data are expressed as the mean ± SD of two experiments. Statistical significance was assessed by the two-sample t test (independent variable). At the end of the experiment, the animals were euthanized and tumors removed and fixed in ten% formaldehyde. Sections were stained with hematoxylin/eosin and for immunohistochemistry equally described below. At the finish of the experiment, subsets of xenograft-begetting mice (northward=four) were injected i.p. with 2-[18F]-fluoro-2-deoxyglucose (18F-FDG, 150μCi, 100 μL in HtwoO) and, afterward 45 min, were anesthetized with 2% isoflurane in oxygen and transferred to a R-4 Rodent Scanner (CTI Concorde Microsystems) micro-positron emission tomograph to capture images. Regions of interest in the tumors and cerebellum were quantified in quadruplicate and are expressed as the hateful ± SD of the ratio of tumor:cerebellar FDG uptake. Animate being experiments were carried out in accordance with established practices as described in the National Institutes of Health Guide for Intendance and Apply of Laboratory Animals and were approved by the Academy of Louisville Institutional Creature Care and Use Committee.
Immunohistochemistry
Five μm mounted sections of formalin-fixed and paraffin-embedded tumor tissue were deparaffinized with xylene. Epitope retrieval was carried out using citrate buffer in a 2100 Retriever (PickCell Laboratories). The sections were blocked with 10% goat serum for 1 hour, then incubated with main antibiotic against PFKFB3, PFKFB4, carbonic anhydrase IX (Proteintech) or cleaved caspase-3 (Prison cell Signaling, Danvers, MA) overnight, followed past an HRP-linked goat anti-rabbit secondary antibody (ane:300, Pierce Biotechnology). The sections were developed with 3,3′-diaminobenzidine tetrahydrochloride (DAB, Vector Laboratories, Burlingame, CA) for 2 min, nuclei counterstained with Mayer'south hematoxylin (Sigma-Aldrich) for 2 min and coverslips attached with Permount (Fisher Scientific, Fair Lawn, NJ). Slides were scanned using a ScanScope XT Digital Slide Scanner (Aperio), data analyzed with the positive pixel count algorithm (ImageScope, Aperio) and a minimum of five fields (20x magnification) were quantified for each tumor section. The information is depicted as % positive pixels/total pixels ± SD.
Acknowledgments
We thank the post-obit funding agencies for their support of these studies: ST, NCI 1R01CA140991 and American Cancer Gild RSG-10-021-01-CNE; and JC, NCI 1R01CA149438 and NCRR CoBRE 1P30GM106396. Nosotros also thank Drs. John Eaton and Otto Grubraw for assisting with the estimation of our data and Drs. Brian Clem and Robert Mitchell for useful discussions.
Footnotes
Editorial annotation:
This paper has been accustomed based in part on peerreview conducted by another journal and the authors' response and revisions as well as expedited peer-review in Oncotarget.
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