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Fructose-2,6-bisphosphate synthesis by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4) is required for the glycolytic response to hypoxia and tumor growth.

Chesney J, Clark J, Klarer AC, Imbert-Fernandez Y, Lane AN, Telang S - Oncotarget (2014)

Bottom Line: The F2,6BP concentration is dictated by four bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) with distinct kinase:phosphatase activities.In this study, we demonstrate that recombinant human PFKFB4 kinase activity is 4.3-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.Taken together, our data indicate that the PFKFB4 expressed in multiple transformed cells and tumors functions to synthesize F2,6BP.

View Article: PubMed Central - PubMed

Affiliation: James Graham Brown Cancer Center, Departments of Medicine (Hematology/Oncology), Pediatrics and Biochemistry and Molecular Biology, University of Louisville, Louisville, KY; These authors contributed equally to this work.

ABSTRACT
Fructose-2,6-bisphosphate (F2,6BP) is a shunt product of glycolysis that allosterically activates 6-phosphofructo-1-kinase (PFK-1) resulting in increased glucose uptake and glycolytic flux to lactate. The F2,6BP concentration is dictated by four bifunctional 6-phosphofructo-2-kinase/fructose-2,6-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, it is not clear whether its kinase or phosphatase activity is required for cancer cell survival. In this study, we demonstrate that recombinant human PFKFB4 kinase activity is 4.3-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 transport chain is not fully operational. Taken together, our data indicate 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 treatment of human cancers.

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Effects of PFKFB4 Inhibition on Cancer Cell Glycolysis, ATP and NADPHThe indicated cell lines were transfected with control siRNA (Ctrl) or two PFKFB4 siRNAs (FB41 and FB42) and glycolysis (3H2O production from [5-3H]glucose), intracellular ATP and NADPH were measured (A). Large T antigen-immortalized, tamoxifen (4OHT)-inducible Pfkfb4−/− lung fibroblasts were exposed to 1 nM and 10 μM 4OHT and analyzed for glycolysis, ATP and NADPH 48 hours later (B). Data are expressed as the mean ± SD of three experiments. * p value <0.01 compared to vehicle or control siRNA.
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Figure 3: Effects of PFKFB4 Inhibition on Cancer Cell Glycolysis, ATP and NADPHThe indicated cell lines were transfected with control siRNA (Ctrl) or two PFKFB4 siRNAs (FB41 and FB42) and glycolysis (3H2O production from [5-3H]glucose), intracellular ATP and NADPH were measured (A). Large T antigen-immortalized, tamoxifen (4OHT)-inducible Pfkfb4−/− lung fibroblasts were exposed to 1 nM and 10 μM 4OHT and analyzed for glycolysis, ATP and NADPH 48 hours later (B). Data are expressed as the mean ± SD of three experiments. * p value <0.01 compared to vehicle or control siRNA.

Mentions: We examined the effect of the two PFKFB4 siRNA molecules and genomic deletion of Pfkfb4 on glycolysis (measured by the production of 3H2O from [5-3H]glucose), ATP and NADPH. We predicted that if the kinase function of PFKFB4 is dominant, then PFKFB4 inhibition would suppress (rather than increase) glycolysis and decrease the concentration of ATP which is produced downstream of PFK-1. We found that both glycolysis and ATP were reduced in all five cell lines examined although the ATP depletion in MCF-7 cells was not statistically significant (Fig. 3A). Although we observed variable effects on NADPH, we did find that PFKFB4 inhibition increased NADPH in H460, A549, LNCaP and MCF-7 cells, presumably as a result of increased availability of F6P for the activity of glucose 6-phosphate isomerase and the oxidative pentose shunt (Fig. 3A). Importantly, we found that Pfkfb4 genomic deletion reduced glycolysis and ATP and increased NADPH, all findings consistent with a requirement of PFKFB4 for the activation of PFK-1 (Fig. 3B).


Fructose-2,6-bisphosphate synthesis by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4) is required for the glycolytic response to hypoxia and tumor growth.

Chesney J, Clark J, Klarer AC, Imbert-Fernandez Y, Lane AN, Telang S - Oncotarget (2014)

Effects of PFKFB4 Inhibition on Cancer Cell Glycolysis, ATP and NADPHThe indicated cell lines were transfected with control siRNA (Ctrl) or two PFKFB4 siRNAs (FB41 and FB42) and glycolysis (3H2O production from [5-3H]glucose), intracellular ATP and NADPH were measured (A). Large T antigen-immortalized, tamoxifen (4OHT)-inducible Pfkfb4−/− lung fibroblasts were exposed to 1 nM and 10 μM 4OHT and analyzed for glycolysis, ATP and NADPH 48 hours later (B). Data are expressed as the mean ± SD of three experiments. * p value <0.01 compared to vehicle or control siRNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196155&req=5

Figure 3: Effects of PFKFB4 Inhibition on Cancer Cell Glycolysis, ATP and NADPHThe indicated cell lines were transfected with control siRNA (Ctrl) or two PFKFB4 siRNAs (FB41 and FB42) and glycolysis (3H2O production from [5-3H]glucose), intracellular ATP and NADPH were measured (A). Large T antigen-immortalized, tamoxifen (4OHT)-inducible Pfkfb4−/− lung fibroblasts were exposed to 1 nM and 10 μM 4OHT and analyzed for glycolysis, ATP and NADPH 48 hours later (B). Data are expressed as the mean ± SD of three experiments. * p value <0.01 compared to vehicle or control siRNA.
Mentions: We examined the effect of the two PFKFB4 siRNA molecules and genomic deletion of Pfkfb4 on glycolysis (measured by the production of 3H2O from [5-3H]glucose), ATP and NADPH. We predicted that if the kinase function of PFKFB4 is dominant, then PFKFB4 inhibition would suppress (rather than increase) glycolysis and decrease the concentration of ATP which is produced downstream of PFK-1. We found that both glycolysis and ATP were reduced in all five cell lines examined although the ATP depletion in MCF-7 cells was not statistically significant (Fig. 3A). Although we observed variable effects on NADPH, we did find that PFKFB4 inhibition increased NADPH in H460, A549, LNCaP and MCF-7 cells, presumably as a result of increased availability of F6P for the activity of glucose 6-phosphate isomerase and the oxidative pentose shunt (Fig. 3A). Importantly, we found that Pfkfb4 genomic deletion reduced glycolysis and ATP and increased NADPH, all findings consistent with a requirement of PFKFB4 for the activation of PFK-1 (Fig. 3B).

Bottom Line: The F2,6BP concentration is dictated by four bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) with distinct kinase:phosphatase activities.In this study, we demonstrate that recombinant human PFKFB4 kinase activity is 4.3-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.Taken together, our data indicate that the PFKFB4 expressed in multiple transformed cells and tumors functions to synthesize F2,6BP.

View Article: PubMed Central - PubMed

Affiliation: James Graham Brown Cancer Center, Departments of Medicine (Hematology/Oncology), Pediatrics and Biochemistry and Molecular Biology, University of Louisville, Louisville, KY; These authors contributed equally to this work.

ABSTRACT
Fructose-2,6-bisphosphate (F2,6BP) is a shunt product of glycolysis that allosterically activates 6-phosphofructo-1-kinase (PFK-1) resulting in increased glucose uptake and glycolytic flux to lactate. The F2,6BP concentration is dictated by four bifunctional 6-phosphofructo-2-kinase/fructose-2,6-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, it is not clear whether its kinase or phosphatase activity is required for cancer cell survival. In this study, we demonstrate that recombinant human PFKFB4 kinase activity is 4.3-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 transport chain is not fully operational. Taken together, our data indicate 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 treatment of human cancers.

Show MeSH
Related in: MedlinePlus