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2006 | 53 | 4 | 789-799
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Hypoxic regulation of PFKFB-3 and PFKFB-4 gene expression in gastric and pancreatic cancer cell lines and expression of PFKFB genes in gastric cancers

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Previously we have shown that hypoxia strongly induces the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 and -4 (PFKFB-3 and PFKFB-4) genes in several cancer cell lines via a HIF-dependent mechanism. In this paper we studied the expression and hypoxic regulation of PFKFB-4 and PFKFB-3 mRNA as well as its correlation with HIF-1α, HIF-2α, VEGF and Glut1 mRNA expression in the pancreatic cancer cell line Panc1 and two gastric cancer cell lines MKN45 and NUGC3. This study clearly demonstrated that PFKFB-3 and PFKFB-4 mRNA are expresses in MKN45, NUGC3 and Panc1 cancers cells and that both genes are responsive to hypoxia in vitro. However, their basal level of expression and hypoxia responsiveness vary in the different cells studied. Particularly, PFKFB-3 mRNA is highly expressed in MKN45 and NUGC3 cancer cells, with the highest response to hypoxia in the NUGC3 cell line. The PFKFB-4 mRNA has a variable low basal level of expression in both gastric and pancreatic cancer cell lines. However, the highest hypoxia response of PFKFB-4 mRNA is found in the pancreatic cancer cell line Panc1. The basal level of PFKFB-4 protein expression is the highest in NUGC3 gastric cancer cell line and lowest in Panc1 cells, with the highest response to hypoxia in the pancreatic cancer cell line. Further studies showed that PFKFB-3 and PFKFB-4 gene expression was highly responsive to the hypoxia mimic dimethyloxalylglycine, a specific inhibitor of HIF-α hydroxylase enzymes, suggesting that the hypoxia responsiveness of PFKFB-3 and PFKFB-4 genes in these cell lines is regulated by the HIF transcription complex. The expression of VEGF and Glut1, which are known HIF-dependent genes, is also strongly induced under hypoxic conditions in gastric and pancreatic cancer cell lines. The levels of HIF-1α protein are increased in both gastric and pancreatic cancer cell lines under hypoxic conditions. However, the basal level of HIF-1α as well as HIF-2α mRNA expression and their hypoxia responsiveness are different in the MKN45 and NUGC3 cancer cells. Thus, the expression of HIF-1α mRNA is decreased in both gastric cancer cell lines treated by hypoxia or dimethyloxalylglycine, but HIF-2α mRNA expression is not changed significantly in NUGC3 and slightly increased in MKN45 cells. Expression of PFKFB-4 and PFKFB-3 was also studied in gastric cancers and corresponding nonmalignant tissue counterparts from the same patients on both the mRNA and protein levels. The expression of PFKFB-3 and PFKFB-4 mRNA as well as PFKFB-1 and PFKFB-2 mRNA was observed in normal human gastric tissue and was increased in malignant gastric tumors. The basal level of PFKFB-4 protein expression in gastric cancers was much higher as compared to the PFKFB-3 isoenzyme. In conclusion, this study provides evidence that PFKFB-4 and PFKFB-3 genes are also expressed in gastric and pancreatic cancer cells, they strongly respond to hypoxia via a HIF-1α dependent mechanism and, together with the expression of PFKFB-1 and PFKFB-2 genes, possibly have a significant role in the Warburg effect which is found in malignant cells.
Physical description
  • Department of Molecular Biology, Palladin Institute of Biochemistry, National Academy of Science of Ukraine, Kyiv, Ukraine
  • Department of Molecular Biology, Palladin Institute of Biochemistry, National Academy of Science of Ukraine, Kyiv, Ukraine
  • Department of Molecular Biology, Palladin Institute of Biochemistry, National Academy of Science of Ukraine, Kyiv, Ukraine
  • INSERM E113 Molecular Mechanisms of Angiogenesis Laboratory, University Bordeaux I, Talence, France
  • Cardeza Foundation for Hematologic Research, Department of Medicine, Jefferson Medical College of Thomas Jefferson University, Philadelphia, PA, USA
  • Investigative Treatment Division, National Cancer Center Research Institute East, Kashiwa, Chiba, Japan
  • Department of Molecular Biology, Palladin Institute of Biochemistry, National Academy of Science of Ukraine, Kyiv, Ukraine
  • Appelhoff RJ, Tian Y-M, Raval RR, Turley H, Harris AL, Pugh CW, Ratcliffe PJ, Gleade JM (2004) Differential function of the prolyl hydroxylases PHD1, PHD2, and PHD3 in the regulation of hypoxia-inducible factor. J Biol Chem 279: 38458-38465.
  • Atsumi T, Chesney J, Metz C, Leng L, Donnelly S, Makita Z, Mitchell R, Bucala R (2002) High expression of inducible 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (iPFK-2; PFKFB-3) in human cancers. Cancer Res 62: 5881-5887.
  • Bando H, Atsumi T, Nishio T, Niwa H, Mishima S, Shimizu C, Yoshioka N, Bucala R, Koike T (2005) Phosphorylation of the 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase/PFKFB-3 family of glycolytic regulators in human cancer. Clin Cancer Res 11: 5784-5792.
  • Bitlon RL, Booker GW (2003) The subtle side to hypoxia inducible factor (HIFα) regulation. Eur J Biochem 270: 791-798.
  • Brown JM, Giaccia AM (1998) The unique physiology of solid tumors: opportunities (and problems) for cancer therapy. Cancer Res 58: 1408-1416.
  • Bruick RK (2003) Oxygen sensing in the hypoxic response pathway: regulation of the hypoxia-inducible transcription factor. Genes Dev 17: 2614-2623.
  • Chesney J, Mitchell R, Benigni F, Bacher M, Spiegel L, Al-Abed Y, Han JH, Metz C, Bucala R (1999) An inducible gene product for 6-phosphofructo-2-kinase with an AU-rich instability element: Role in tumor cell glycolysis and the Warburg effect. Proc Natl Acad Sci USA 96: 3047-3052.
  • Dang CV, Semenza GL (1999) Oncogenic alterations of metabolism. Trends Biochem Sci 24: 68-76.
  • Depping R, Hagele S, Wagner KF, Wiesner RJ, Camenisch G, Wenger RH, Katschinski DM (2004) A dominant-negative isoform of hypoxia-inducible factor-1α specifically expressed in human testis. Biol Reprod 71: 331-339.
  • Elvidge GP, Glenny L, Appelhoff RJ, Ratcliffe PJ, Ragoussis J, Gleadle JM (2006) Concordant regulation of gene expression by hypoxia and 2-oxoglutarate dependent dioxygenase inhibition; the role of HIF-1α, HIF-2α and other pathways. J Biol Chem 281: 15215-15226.
  • Epstein AC, Gleadle JM, McNeill LA, Hewitson KS, O'Rourke J, Mole DR, Mukherji M, Metzen E, Wilson MI, Dhanda A, Tian YM, Masson N, Hamilton DL, Jaakkola P, Barstead R, Hodgkin J, Maxwell PH, Pugh CW, Schofield CJ, Ratcliffe PJ (2001) C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 107: 43-54.
  • Fukasawa M, Tsuchiya T, Takayama E, Shinomiya N, Uyeda K, Sakakibara R, Seki S (2004) Identification and characterization of the hypoxia-responsive element of the human placental 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene. J Biochem (Tokyo) 136: 273-277.
  • Gleade JM, Ratcliffe PJ (1998) Hypoxia and the regulation of gene expression. Mol Med Today 4: 122-129.
  • Gleadle JM, Ebert BL, Firth JD, Ratcliffe PJ (1995) Regulation of angiogenic growth factor expression by hypoxia, transition metals, and chelating agents. Am J Physiol 268: C1362-C1368.
  • Goyal P, Weissmann N, Grimminger F, Hegel C, Bader L, Rose F, Fink L, Ghofrani HA, Schermuly RT, Schmidt HH, Seeger W, Hanze J (2004) Upregulation of NAD(P)H oxidase 1 in hypoxia activates hypoxia-inducible factor 1 via increase in reactive oxygen species. Free Radic Biol Med 36: 1279-1288.
  • Greijer AE, van der Groep P, Kemming D, Shvarts A, Semenza GL, Meijer GA, van de Wiel MA, Belien JA, van Diest PJ, van der Wall E (2005) Up-regulation of gene expression by hypoxia is mediated predominantly by hypoxia-inducible factor 1 (HIF-1). J Pathol 206: 291-304.
  • Hockel M, Vaupel P (2001) Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects. J Natl Cancer Inst 93: 266-276.
  • Hopfl G, Ogunshola O, Gassmann M (2004) HIFs and tumors - causes and consequences. Am J Physiol Regul Integr Comp Physiol 286: R608-R623.
  • Hu CJ, Wang LY, Chodosh LA, Keith B, Simon MC (2003) Differential roles of hypoxia-inducible factor 1α (HIF-1α) and HIF-2α in hypoxic gene regulation. Mol Cell Biol 23: 9361-9374.
  • Huang LE, Bunn HF (2003) Hypoxia-inducible factor and its biomedical relevance. J Biol Chem 278: 19575-19578.
  • Hue L, Beauloye C, Bertrand L, Horman S, Krause U, Marsin A-S, Meisse D, Vertommen D, Rider MH (2003) New targets of AMP-activated protein kinase. Biochem Soc Trans 31: 213-215.
  • Ivan M, Kondo K, Yang H, Kim W, Valiando J, Ohh M, Salic A, Asara JM, Lane WS, Kaelin WGJ (2001) HIFα targeted for VHL-mediated destruction by proline hydroxylation: implications for O2> sensing. Science 292: 464-468.
  • Jaakkola P, Mole DR, Tian Y-M, Wilson MI, Gielbert J, Gaskell SJ, von Kriegsheim A, Hebestreit HF, Mukherji M, Schofield CJ, Maxwell PH, Pugh CW, Ratcliffe PJ (2001) Targeting of HIF-α to the von Hippel-Lindau ubiquitylation complex by O2>-regulated prolyl hydroxylation. Science 292: 468-472.
  • Kim SG, Manes NP, El-Maghrabi MR, Lee YH (2006) Crystal structure of the hypoxia-inducible form of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3): a possible new target for cancer therapy. J Biol Chem 281: 2939-2944.
  • Lando D, Peet DJ, Whelan DA, Gorman JJ, Whitelaw ML (2002) Asparagine hydroxylation of the HIF transactivation domain: a hypoxic switch. Science 295: 858-861.
  • Lu H, Forbes RA, Verma A (2002) Hypoxia-inducible factor 1 activation by aerobic glycolysis implicates the Warburg effect in carcinogenesis. J Biol Chem 277: 23111-23115.
  • Makino Y, Cao RH, Svensson K, Bertilsson GR, Asman M, Tanaka H, Cao YH, Berkenstam A, Poellinger L (2001) Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression. Nature 414: 550-554.
  • Makino Y, Kanopka A, Wilson WJ, Tanaka H, Poellinger L (2002) Inhibitory PAS domain protein (IPAS) is a hypoxia-inducible splicing variant of the hypoxia-inducible factor-3α locus. J Biol Chem 277: 32405-32408.
  • Manalo DJ, Rowan A, Lavoie T, Natarajan L, Kelly BD, Ye SQ, Garcia JG, Semenza GL (2005) Transcriptional regulation of vascular endothelial cell responses to hypoxia by HIF-1. Blood 105: 659-669.
  • Manzano A, Rosa JL, Ventura F, Perez JX, Nadal M, Estivill X, Ambrosio S, Gil J, Bartrons R (1998) Cloning, expression and chromosomal localization of a human testis 6-phosphofructo-2-kinase/fructose-2,6-bisphospha­tase gene. Gene 229: 83-89.
  • Marsin AS, Douzin C, Bertrand L, Hue L (2002) The stimulation of glycolysis by hypoxia in activated monocytes is mediated by AMP-activated protein kinase and inducible 6-phosphofructo-2-kinase. J Biol Chem 277: 30778-30783.
  • Masson N, Ratcliffe PJ (2003) HIF prolyl and asparaginyl hydroxylases in the biological response to intracellular O2> levels. J Cell Sci 116: 3041-3049.
  • Metzen E, Ratcliffe PJ (2004) HIF hydroxylation and cellular oxygen sensing. Biol Chem 385: 223-230.
  • Min J-H, Yang H, Ivan M, Gertler F, Kaelin WGJ Jr, Pavletich NP (2002) Structure of an HIF-1α-pVHL complex: hydroxyproline recognition in signaling. Science 296: 1886-1889.
  • Minchenko A, Caro J (2000) Regulation of endothelin-1 gene expression in human microvascular endothelial cells by hypoxia and cobalt: role of hypoxia responsive element. Mol Cell Biochem 208: 53-62.
  • Minchenko A, Bauer T, Salceda S, Caro J (1994) Hypoxic stimulation of vascular endothelial growth factor expression in vitro and in vivo. Lab Invest 71: 374-379.
  • Minchenko A, Leshchinsky I, Opentanova I, Sang N, Srinivas V, Armstead V, Caro J (2002) Hypoxia-inducible factor-1-mediated expression of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) gene: Its possible role in the Warburg effect. J Biol Chem 277: 6183-6187.
  • Minchenko O, Opentanova I, Caro J (2003) Hypoxic regulation of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene family (PFKFB-1-4) expression in vivo. FEBS Lett 554: 264-270.
  • Minchenko OH, Opentanova IL, Minchenko DO, Ogura T, Esumi H (2004) Hypoxia induces transcription of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-4 gene via hypoxia inducible factor-1α activation. FEBS Lett 576: 14-20.
  • Minchenko OH, Ochiai A, Opentanova IL, Ogura T, Minchenko DO, Caro J, Komisarenko SV, Esumi H (2005a) Overexpression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-4 in the human breast and colon malignant tumors. Biochimie 87: 1005-1010.
  • Minchenko OH, Ogura T, Opentanova IL, Minchenko DO, Ochiai A, Caro J, Komisarenko SV, Esumi H (2005b) 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene family overexpression in the lung tumor. Ukr Biokhim Zh 77: 46-50.
  • Minchenko OH, Opentanova IL, Ogura T, Minchenko DO, Komisarenko SV, Caro J, Esumi H (2005c) Expression and hypoxia-responsiveness of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 in the mammary gland malignant cell lines. Acta Biochim Polon 52: 881-888.
  • Mole DR, Schlemminger I, McNeil LA, Hewitson KS, Pugh CW, Ratcliffe PJ, Schofield CJ (2003) 2-Oxoglutarate analogue inhibitors of HIF prolyl hydroxylase. Bioorg Med Chem Lett 13: 2677-2680.
  • Obach M, Navarro-Sabate A, Caro J, Kong X, Duran J, Gomez M, Perales JC, Ventura F, Rosa JL, Bartrons R (2004) 6-Phosphofructo-2-kinase (pfkfb3) gene promoter contains hypoxia-inducible factor-1 binding sites necessary for transactivation in response to hypoxia. J Biol Chem 279: 53562-53570.
  • Okar DA, Manzano A, Navarro-Sabate A, Riera L, Bartrons R, Lange A (2001) PFK-2/FBPase-2: maker and breaker of the essential biofactor fructose-2,6-bisphosphate. Trends Biochem Sci 26: 30-35.
  • Pilkis SJ, Claus TH, Kurland IJ, Lange AJ (1995) 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase: a metabolic signaling enzyme. Annu Rev Biochem 64: 799-835.
  • Ratcliffe PJ, O'Rourke JF, Maxwell PH, Pugh CW (1998) Oxygen sensing, hypoxia-inducible factor-1 and the regulation of mammalian gene expression. J Exp Biol 201: 1153-1162.
  • Rider MH, Bertrand L, Vertommen D, Michels PA, Rousseau GG, Hue L (2004) 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase: head-head with a bifunctional enzyme that controls glycolysis. Biochem J 381: 561-578.
  • Sakakibara R, Okudaira T, Fujiwara K, Kato M, Hirata T, Yamanaka S, Naito M, Fukasawa M (1999) Tissue distribution of placenta-type 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Biochem Biophys Res Commun 257: 177-181.
  • Sakata J, Abe Y, Uyeda K (1991) Molecular cloning of the DNA and expression and characterization of rat testes fructose-6-phosphate-2-kinase:fructose-2,6-bisphosphatase. J Biol Chem 266: 15764-15770.
  • Schofield CJ, Ratcliffe PJ (2004) Oxygen sensing by HIF hydroxylases. Nat Rev Mol Cell Biol 5: 343-354.
  • Seagroves TN, Ryan HE, Lu H, Wouters BG, Knapp M, Thibault P, Laderoute K, Johnson RS (2000) Transcription factor HIF-1 is a necessary mediator of the Pasteur effect in mammalian cells. Mol Cell Biol 21: 3436-3444.
  • Semenza GL (2000) HIF-1: mediator of physiological and pathophysiological responses to hypoxia. J Appl Physiol 88: 1474-1480.
  • Semenza GL (2001) HIF-1, O(2), and the 3 PHDs: how animal cells signal hypoxia to the nucleus. Cell 107: 1-3.
  • Semenza GL (2002) Involvement of hypoxia-inducible factor 1 in human cancer. Intern Med 41: 79-83.
  • Sowter HM, Raval RR, Moore JW, Ratcliffe PJ, Harris AL (2003) Predominant role of hypoxia-inducible transcription factor (Hif)-1α versus Hif-2α in regulation of the transcriptional response to hypoxia. Cancer Res 63: 6130-6134.
  • Stoeltzing O, McCarty MF, Wey JS, Fan F, Liu W, Belcheva A, Bucana CD, Semenza GL, Ellis LM (2004) Role of hypoxia-inducible factor 1α in gastric cancer cell growth, angiogenesis, and vessel maturation. J Natl Cancer Inst 96: 946-956.
  • Vaupel P (1996) Oxygen transport in tumors: characteristics and clinical implications. Adv Exp Med Biol 388: 341-351.
  • Wenger RH (2002) Cellular adaptation to hypoxia: O2>-sensing protein hydroxylases, hypoxia-inducible transcription factors, and O2>-regulated gene expression. FASEB J 16: 1151-1162.
  • Wykoff CC, Pugh CW, Harris AL, Maxwell PH, Ratcliffe PJ (2001) The HIF pathway: implications for patterns of gene expression in cancer. Novartis Found Symp 240: 212-225.
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