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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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PFKFB4 Is Expressed in Multiple Normal Tissues and Over-Expressed in Human Lung AdenocarcinomasPFKFB1-4 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 by multiplex RT-PCR (B) and by real-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 mean ± SD of three experiments. *p value < 0.001 compared to normal tissue controls.
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Figure 1: PFKFB4 Is Expressed in Multiple Normal Tissues and Over-Expressed in Human Lung AdenocarcinomasPFKFB1-4 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 by multiplex RT-PCR (B) and by real-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 mean ± SD of three experiments. *p value < 0.001 compared to normal tissue controls.

Mentions: We first conducted a simultaneous analysis of PFKFB1-4 mRNA expression in normal human tissues using real-time RT-PCR for each family member. We observed high expression of: PFKFB1 in liver and skeletal muscle; PFKFB2 in heart, 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 as being inducible [18] and ubiquitous [19], was expressed at very low levels in all tissues with the exception of leukocytes (Fig 1A). We then used multiplex RT-PCR and real-time RT-PCR to assess PFKFB1-4 mRNA expression in normal lung tissues and adjacent lung adenocarcinomas from five patients. Only PFKFB4 mRNA was over-expressed in each of these patients’ tumors (Fig. 1B, C). Further examination 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, E).


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)

PFKFB4 Is Expressed in Multiple Normal Tissues and Over-Expressed in Human Lung AdenocarcinomasPFKFB1-4 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 by multiplex RT-PCR (B) and by real-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 mean ± SD of three experiments. *p value < 0.001 compared to normal tissue controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: PFKFB4 Is Expressed in Multiple Normal Tissues and Over-Expressed in Human Lung AdenocarcinomasPFKFB1-4 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 by multiplex RT-PCR (B) and by real-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 mean ± SD of three experiments. *p value < 0.001 compared to normal tissue controls.
Mentions: We first conducted a simultaneous analysis of PFKFB1-4 mRNA expression in normal human tissues using real-time RT-PCR for each family member. We observed high expression of: PFKFB1 in liver and skeletal muscle; PFKFB2 in heart, 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 as being inducible [18] and ubiquitous [19], was expressed at very low levels in all tissues with the exception of leukocytes (Fig 1A). We then used multiplex RT-PCR and real-time RT-PCR to assess PFKFB1-4 mRNA expression in normal lung tissues and adjacent lung adenocarcinomas from five patients. Only PFKFB4 mRNA was over-expressed in each of these patients’ tumors (Fig. 1B, C). Further examination 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, E).

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