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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: 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.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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Role of PFKFB3 and PFKFB4 in Hypoxia-Induced F2,6BP production, Glucose Uptake, Glycolysis, ATP and NADPHH460 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 blot analysis and densitometry (B and C), F2,6BP (D), 14C-2-DG uptake (E), glycolysis (3H2O production from [5-3H]glucose) (F), intracellular ATP (G) and NADPH (H). Data are expressed as the mean ± SD of three experiments. * p value < 0.01 compared to control siRNA.
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Figure 6: Role of PFKFB3 and PFKFB4 in Hypoxia-Induced F2,6BP production, Glucose Uptake, Glycolysis, ATP and NADPHH460 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 blot analysis and densitometry (B and C), F2,6BP (D), 14C-2-DG uptake (E), glycolysis (3H2O production from [5-3H]glucose) (F), intracellular ATP (G) and NADPH (H). Data are expressed as the mean ± SD of three experiments. * p value < 0.01 compared to control siRNA.

Mentions: Both PFKFB3 and PFKFB4 mRNA previously have been found to be induced by hypoxia [10, 12, 13, 22-24] and we confirmed these past observations in H460 cells (Fig. 6A). In order to compare the functional relevance of PFKFB3 and PFKFB4 under hypoxic conditions, we transfected H460 cells with PFKFB4 or PFKFB3 siRNA under 21% and 1% oxygen. We observed a reduction in the hypoxic induction of both enzyme mRNAs as a result of the siRNA molecules (Fig. 6A). Hypoxia strongly induced PFKFB4 protein expression, as has been observed in other cell lines (Fig. 6A-C). Interestingly, we found a far lower induction of PFKFB3 protein which may already be maximally expressed as a result of the multitude of genetic alterations that increase PFKFB3 protein expression such as PTEN loss and Ras activation (e.g. H460 cells express KRASQ61H) [20, 25, 26]. Importantly, transfection with either PFKFB4 or PFKFB3 siRNA caused 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).


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)

Role of PFKFB3 and PFKFB4 in Hypoxia-Induced F2,6BP production, Glucose Uptake, Glycolysis, ATP and NADPHH460 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 blot analysis and densitometry (B and C), F2,6BP (D), 14C-2-DG uptake (E), glycolysis (3H2O production from [5-3H]glucose) (F), intracellular ATP (G) and NADPH (H). Data are expressed as the mean ± SD of three experiments. * p value < 0.01 compared to control siRNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Role of PFKFB3 and PFKFB4 in Hypoxia-Induced F2,6BP production, Glucose Uptake, Glycolysis, ATP and NADPHH460 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 blot analysis and densitometry (B and C), F2,6BP (D), 14C-2-DG uptake (E), glycolysis (3H2O production from [5-3H]glucose) (F), intracellular ATP (G) and NADPH (H). Data are expressed as the mean ± SD of three experiments. * p value < 0.01 compared to control siRNA.
Mentions: Both PFKFB3 and PFKFB4 mRNA previously have been found to be induced by hypoxia [10, 12, 13, 22-24] and we confirmed these past observations in H460 cells (Fig. 6A). In order to compare the functional relevance of PFKFB3 and PFKFB4 under hypoxic conditions, we transfected H460 cells with PFKFB4 or PFKFB3 siRNA under 21% and 1% oxygen. We observed a reduction in the hypoxic induction of both enzyme mRNAs as a result of the siRNA molecules (Fig. 6A). Hypoxia strongly induced PFKFB4 protein expression, as has been observed in other cell lines (Fig. 6A-C). Interestingly, we found a far lower induction of PFKFB3 protein which may already be maximally expressed as a result of the multitude of genetic alterations that increase PFKFB3 protein expression such as PTEN loss and Ras activation (e.g. H460 cells express KRASQ61H) [20, 25, 26]. Importantly, transfection with either PFKFB4 or PFKFB3 siRNA caused 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).

Bottom Line: 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.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