Final Report Summary - FP2ONCO (ROLE OF PFKFB3 IN THE REGULATION OF THE CELL CYCLE AND TUMORIGENESIS)
Cancer is a leading cause of death in the western world, second only to cardiovascular disease, and is therefore a European public health problem of overwhelming human and economic significance. Understanding the precise molecular mechanisms leading to oncogenic transformation and cancer progression is essential to fighting cancer effectively, as this helps us design better therapeutic approaches targeted at curing cancer.
Fast proliferating cells, e.g. tumor cells, must be able to tightly control and coordinate glucose metabolism and the cell cycle in order to guarantee sufficient ATP and anabolic substrates at distinct phases of the cell cycle. Fructose-2,6-bisphosphate (F2,6BP) is a potent activator of 6-phosphofructo kinase (PFK1), one of the rate-limiting enzymes of glycolysis1. The steady-state intracellular concentration of F2,6BP is set by the family of enzymes known as 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB), which are encoded by four genes (PFKFB1-4)2. The protein product of the PFKFB3 gene (PFKFB3) is overexpressed in tumors3 and required for ras-induced tumorigenesis4, and inhibition of PFKFB3 results in suppression of tumor growth in vivo5. Although most functional studies of the role of PFKFB3 in cancer progression have invoked its well-recognized function in the regulation of glycolysis, recent observations have established that PFKFB3 also traffics to the nucleus and that its product, F2,6BP, induces cellular proliferation in the nucleus6. However, the role of endogenous PFKFB3 in regulation of the cell cycle and cell survival remains elusive. In this study, HeLa cells (Human cervical cancer cell line) as a model system was used in order to determine the role of PFKFB3 in the cell cycle and cell survival. Results were also confirmed in the human colon cancer cell line HCT116.
We started out by confirming the requirement of endogenous PFKFB3 in the glycolytic flux of HeLa cells. HeLa cells were transfected with two separate siRNA molecules that were specific to the PFKFB3 mRNA. 48 h later, we found that PFKFB3 siRNA molecules potently reduced F2,6BP, glucose uptake and glycolysis. We next examined the effects of PFKFB3 silencing on exponentially growing HeLa cells over 24-72 h and observed a complete suppression of cell proliferation after 48 and 72 h. Interestingly, this antiproliferative effect far exceeded the antiglycolytic effect. We then went on to show that the reduced viable cell number in PFKFB3-depleted cells was due to a combination of a G1/S transition block and by an increase in early and late apoptotic cells as assessed by flow cytometry. In our prior study6, we had found that PFKFB3 traffics to the nucleus and the nuclear PFKFB3 suppresses p27 protein expression. We then went on to show that the effect of PFKFB3 on the p27 protein is mediated by stimulation of Cdk1 activity by F2,6BP produced in the nucleus. p27 is a non-classical tumor suppressor, and is both an inhibitor and substrate of Cdk1. Phosphorylation of p27 at threonine 187 by Cdk1 marks the p27 protein for proteasomal degradation7. Based on these studies, we speculated that PFKFB3 inhibition would have the opposite effect and thus suppresses Cdk activity. To this end, PFKFB3 was silenced in HeLa cells and 48 h alter, p27 mRNA and protein levels were analysed by real-time RT-PCR and Western blot, respectively. We found that PFKFB3 silencing caused a significant increase in the p27 protein, whereas no effect was observed on the p27 mRNA, suggesting that the effect of PFKFB3 on p27 expression was posttranscriptional as expected. We then immunoprecipitated Cdk1 and an in vitro kinase assay was performed using recombinant p27 protein as substrate. We observed a marked reduction in immunoprecipitated Cdk1 activity in PFKFB3-depleted cells compared with control cells that correlated with the reduction in cell cycle progression and apoptosis. We next measured in situ Cdk1 activity by assessing the phosphorylation of serines within the Cdk1-specific (K/R)(S*)PX(K/R) motif. We observed a reduction in the phosphorylation of Cdk1 serine substrates in both the cytoplasm and nucleus after PFKFB3 siRNA transfection. One such Cdk1 substrate, Cdh1, binds to and activates the APC/C ubiquitin ligase8 – this activation is attenuated by Cdk-mediated phosphorylation of Cdh1. We therefore postulated that PFKFB3 inhibition might reduce Cdh1 phosphorylation, which in turn might increase APC/C-Cdh1 activity and decrease the stability of its substrates, which include PFKFB3 and cyclin B18. We found that PFKFB3 silencing did in fact reduce phospho-Cdh1, which in turn led to reduced levels of cyclin B1. However, precise mechanisms by which the product of PFKFB3, F2,6BP, activates Cdk1 remain unknown. However, we went on to show that in addition to cyclin B1, cyclin D3 levels were also markedly decreased in PFKFB3-depleted cells. It remains to be determined whether the decreased cyclin levels in PFKFB3-depleted cells contributes the observed decrease in immunoprecipitated Cdk1 activity.
Given that PFKFB3 inhibition reduces Cdk1 activity and stabilizes p27 and because p27 is a potent suppressor of G1/S transition and inducer of apoptosis9, we postulated that p27 may itself be mediating the pro-apoptotic and cytostatic effects. In order to assess the role of p27 in mediating the apoptotic and cytostatic effects of PFKFB3 inhibition, we transfected HeLa cells with control siRNA, PFKFB3 siRNA, p27 siRNA or both the PFKFB3 and p27 siRNA molecules and analyzed the effects on cell cycle and apoptosis. We found that p27 siRNA transfection resulted in near complete reversal of the G1/S transition block and induction of apoptosis. These data demonstrate for the first time that p27 expression is required for the antiproliferative and pro-apoptotic effects of PFKFB3 inhibition.
A small molecule antagonist of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), was recently identified using a combination of computational modeling and receptor-based in silico screening5. 3PO and more potent 3PO derivatives have antitumor properties and can stimulate the apoptosis of transformed cells. We examined LLC cells after 3PO exposure and LLCs grown as subcutaneous tumors in syngeneic mice without and with 3PO exposure and assessed p27 expression by western blot analysis and immunohistochemistry, respectively. We observed a marked increase in the p27 protein after 3PO exposure in vitro and in vivo. These data indicate that p27 protein expression by tumor cells may be useful as a pharmacodynamic endpoint for incorporation into upcoming clinical trials of PFKFB3 inhibitors.
Homozygous genomic deletion of the Pfkfb3 gene in mice results in embryonic lethality10. Given that p27 silencing reverses the anti-proliferative and apoptotic phenotype caused by PFKFB3 silencing, we speculated that genomic co-deletion of Cdkn1b gene, which encodes p27, may reverse the embryonic lethality phenotype induced by homozygous deletion of the PFKFB3 gene. In order to address this question, mice that are heterozygous for both Pfkfb3 (Pfkfb3+/-) and p27 (Cdkn1b+/-) genes were crossed and an F1 generation that are double heterozygous (Pfkfb3+/-;Cdkn1b+/-) was established. However, interbreeding of sufficient number of F1 did not produce any viable mice that are homozygous knockout for Pfkfb3, suggesting that Pfkfb3 serves a function in embryonic development that is independent of its anti-p27 effect observed in transformed cells.
1 Chesney, J. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and tumor cell glycolysis. Current opinion in clinical nutrition and metabolic care 9, 535-539, doi:10.1097/01.mco.0000241661.15514.fb (2006).
2 Yalcin, A., Telang, S., Clem, B. & Chesney, J. Regulation of glucose metabolism by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases in cancer. Experimental and molecular pathology 86, 174-179, doi:10.1016/j.yexmp.2009.01.003 (2009).
3 Atsumi, T. et al. High expression of inducible 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (iPFK-2; PFKFB3) in human cancers. Cancer research 62, 5881-5887 (2002).
4 Telang, S. et al. Ras transformation requires metabolic control by 6-phosphofructo-2-kinase. Oncogene 25, 7225-7234, doi:10.1038/sj.onc.1209709 (2006).
5 Clem, B. F. et al. Targeting 6-Phosphofructo-2-Kinase (PFKFB3) as a Therapeutic Strategy against Cancer. Molecular cancer therapeutics, doi:10.1158/1535-7163.MCT-13-0097 (2013).
6 Yalcin, A. et al. Nuclear targeting of 6-phosphofructo-2-kinase (PFKFB3) increases proliferation via cyclin-dependent kinases. The Journal of biological chemistry 284, 24223-24232, doi:10.1074/jbc.M109.016816 (2009).
7 Grimmler, M. et al. Cdk-inhibitory activity and stability of p27Kip1 are directly regulated by oncogenic tyrosine kinases. Cell 128, 269-280, doi:10.1016/j.cell.2006.11.047 (2007).
8 Tudzarova, S. et al. Two ubiquitin ligases, APC/C-Cdh1 and SKP1-CUL1-F (SCF)-beta-TrCP, sequentially regulate glycolysis during the cell cycle. Proceedings of the National Academy of Sciences of the United States of America 108, 5278-5283, doi:10.1073/pnas.1102247108 (2011).
9 Kawana, H. et al. Role of p27Kip1 and cyclin-dependent kinase 2 in the proliferation of non-small cell lung cancer. The American journal of pathology 153, 505-513 (1998).
10 Chesney, J. et al. Targeted disruption of inducible 6-phosphofructo-2-kinase results in embryonic lethality. Biochemical and biophysical research communications 331, 139-146, doi:10.1016/j.bbrc.2005.02.193 (2005).
Fast proliferating cells, e.g. tumor cells, must be able to tightly control and coordinate glucose metabolism and the cell cycle in order to guarantee sufficient ATP and anabolic substrates at distinct phases of the cell cycle. Fructose-2,6-bisphosphate (F2,6BP) is a potent activator of 6-phosphofructo kinase (PFK1), one of the rate-limiting enzymes of glycolysis1. The steady-state intracellular concentration of F2,6BP is set by the family of enzymes known as 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB), which are encoded by four genes (PFKFB1-4)2. The protein product of the PFKFB3 gene (PFKFB3) is overexpressed in tumors3 and required for ras-induced tumorigenesis4, and inhibition of PFKFB3 results in suppression of tumor growth in vivo5. Although most functional studies of the role of PFKFB3 in cancer progression have invoked its well-recognized function in the regulation of glycolysis, recent observations have established that PFKFB3 also traffics to the nucleus and that its product, F2,6BP, induces cellular proliferation in the nucleus6. However, the role of endogenous PFKFB3 in regulation of the cell cycle and cell survival remains elusive. In this study, HeLa cells (Human cervical cancer cell line) as a model system was used in order to determine the role of PFKFB3 in the cell cycle and cell survival. Results were also confirmed in the human colon cancer cell line HCT116.
We started out by confirming the requirement of endogenous PFKFB3 in the glycolytic flux of HeLa cells. HeLa cells were transfected with two separate siRNA molecules that were specific to the PFKFB3 mRNA. 48 h later, we found that PFKFB3 siRNA molecules potently reduced F2,6BP, glucose uptake and glycolysis. We next examined the effects of PFKFB3 silencing on exponentially growing HeLa cells over 24-72 h and observed a complete suppression of cell proliferation after 48 and 72 h. Interestingly, this antiproliferative effect far exceeded the antiglycolytic effect. We then went on to show that the reduced viable cell number in PFKFB3-depleted cells was due to a combination of a G1/S transition block and by an increase in early and late apoptotic cells as assessed by flow cytometry. In our prior study6, we had found that PFKFB3 traffics to the nucleus and the nuclear PFKFB3 suppresses p27 protein expression. We then went on to show that the effect of PFKFB3 on the p27 protein is mediated by stimulation of Cdk1 activity by F2,6BP produced in the nucleus. p27 is a non-classical tumor suppressor, and is both an inhibitor and substrate of Cdk1. Phosphorylation of p27 at threonine 187 by Cdk1 marks the p27 protein for proteasomal degradation7. Based on these studies, we speculated that PFKFB3 inhibition would have the opposite effect and thus suppresses Cdk activity. To this end, PFKFB3 was silenced in HeLa cells and 48 h alter, p27 mRNA and protein levels were analysed by real-time RT-PCR and Western blot, respectively. We found that PFKFB3 silencing caused a significant increase in the p27 protein, whereas no effect was observed on the p27 mRNA, suggesting that the effect of PFKFB3 on p27 expression was posttranscriptional as expected. We then immunoprecipitated Cdk1 and an in vitro kinase assay was performed using recombinant p27 protein as substrate. We observed a marked reduction in immunoprecipitated Cdk1 activity in PFKFB3-depleted cells compared with control cells that correlated with the reduction in cell cycle progression and apoptosis. We next measured in situ Cdk1 activity by assessing the phosphorylation of serines within the Cdk1-specific (K/R)(S*)PX(K/R) motif. We observed a reduction in the phosphorylation of Cdk1 serine substrates in both the cytoplasm and nucleus after PFKFB3 siRNA transfection. One such Cdk1 substrate, Cdh1, binds to and activates the APC/C ubiquitin ligase8 – this activation is attenuated by Cdk-mediated phosphorylation of Cdh1. We therefore postulated that PFKFB3 inhibition might reduce Cdh1 phosphorylation, which in turn might increase APC/C-Cdh1 activity and decrease the stability of its substrates, which include PFKFB3 and cyclin B18. We found that PFKFB3 silencing did in fact reduce phospho-Cdh1, which in turn led to reduced levels of cyclin B1. However, precise mechanisms by which the product of PFKFB3, F2,6BP, activates Cdk1 remain unknown. However, we went on to show that in addition to cyclin B1, cyclin D3 levels were also markedly decreased in PFKFB3-depleted cells. It remains to be determined whether the decreased cyclin levels in PFKFB3-depleted cells contributes the observed decrease in immunoprecipitated Cdk1 activity.
Given that PFKFB3 inhibition reduces Cdk1 activity and stabilizes p27 and because p27 is a potent suppressor of G1/S transition and inducer of apoptosis9, we postulated that p27 may itself be mediating the pro-apoptotic and cytostatic effects. In order to assess the role of p27 in mediating the apoptotic and cytostatic effects of PFKFB3 inhibition, we transfected HeLa cells with control siRNA, PFKFB3 siRNA, p27 siRNA or both the PFKFB3 and p27 siRNA molecules and analyzed the effects on cell cycle and apoptosis. We found that p27 siRNA transfection resulted in near complete reversal of the G1/S transition block and induction of apoptosis. These data demonstrate for the first time that p27 expression is required for the antiproliferative and pro-apoptotic effects of PFKFB3 inhibition.
A small molecule antagonist of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), was recently identified using a combination of computational modeling and receptor-based in silico screening5. 3PO and more potent 3PO derivatives have antitumor properties and can stimulate the apoptosis of transformed cells. We examined LLC cells after 3PO exposure and LLCs grown as subcutaneous tumors in syngeneic mice without and with 3PO exposure and assessed p27 expression by western blot analysis and immunohistochemistry, respectively. We observed a marked increase in the p27 protein after 3PO exposure in vitro and in vivo. These data indicate that p27 protein expression by tumor cells may be useful as a pharmacodynamic endpoint for incorporation into upcoming clinical trials of PFKFB3 inhibitors.
Homozygous genomic deletion of the Pfkfb3 gene in mice results in embryonic lethality10. Given that p27 silencing reverses the anti-proliferative and apoptotic phenotype caused by PFKFB3 silencing, we speculated that genomic co-deletion of Cdkn1b gene, which encodes p27, may reverse the embryonic lethality phenotype induced by homozygous deletion of the PFKFB3 gene. In order to address this question, mice that are heterozygous for both Pfkfb3 (Pfkfb3+/-) and p27 (Cdkn1b+/-) genes were crossed and an F1 generation that are double heterozygous (Pfkfb3+/-;Cdkn1b+/-) was established. However, interbreeding of sufficient number of F1 did not produce any viable mice that are homozygous knockout for Pfkfb3, suggesting that Pfkfb3 serves a function in embryonic development that is independent of its anti-p27 effect observed in transformed cells.
1 Chesney, J. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and tumor cell glycolysis. Current opinion in clinical nutrition and metabolic care 9, 535-539, doi:10.1097/01.mco.0000241661.15514.fb (2006).
2 Yalcin, A., Telang, S., Clem, B. & Chesney, J. Regulation of glucose metabolism by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases in cancer. Experimental and molecular pathology 86, 174-179, doi:10.1016/j.yexmp.2009.01.003 (2009).
3 Atsumi, T. et al. High expression of inducible 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (iPFK-2; PFKFB3) in human cancers. Cancer research 62, 5881-5887 (2002).
4 Telang, S. et al. Ras transformation requires metabolic control by 6-phosphofructo-2-kinase. Oncogene 25, 7225-7234, doi:10.1038/sj.onc.1209709 (2006).
5 Clem, B. F. et al. Targeting 6-Phosphofructo-2-Kinase (PFKFB3) as a Therapeutic Strategy against Cancer. Molecular cancer therapeutics, doi:10.1158/1535-7163.MCT-13-0097 (2013).
6 Yalcin, A. et al. Nuclear targeting of 6-phosphofructo-2-kinase (PFKFB3) increases proliferation via cyclin-dependent kinases. The Journal of biological chemistry 284, 24223-24232, doi:10.1074/jbc.M109.016816 (2009).
7 Grimmler, M. et al. Cdk-inhibitory activity and stability of p27Kip1 are directly regulated by oncogenic tyrosine kinases. Cell 128, 269-280, doi:10.1016/j.cell.2006.11.047 (2007).
8 Tudzarova, S. et al. Two ubiquitin ligases, APC/C-Cdh1 and SKP1-CUL1-F (SCF)-beta-TrCP, sequentially regulate glycolysis during the cell cycle. Proceedings of the National Academy of Sciences of the United States of America 108, 5278-5283, doi:10.1073/pnas.1102247108 (2011).
9 Kawana, H. et al. Role of p27Kip1 and cyclin-dependent kinase 2 in the proliferation of non-small cell lung cancer. The American journal of pathology 153, 505-513 (1998).
10 Chesney, J. et al. Targeted disruption of inducible 6-phosphofructo-2-kinase results in embryonic lethality. Biochemical and biophysical research communications 331, 139-146, doi:10.1016/j.bbrc.2005.02.193 (2005).