Skip to main content

Advertisement

SpringerLink
Log in

Intracellular NAD(H) levels control motility and invasion of glioma cells

  • Research Article
  • Published:
Cellular and Molecular Life Sciences Aims and scope Submit manuscript

Abstract

Oncogenic transformation involves reprogramming of cell metabolism, whereby steady-state levels of intracellular NAD+ and NADH can undergo dramatic changes while ATP concentration is generally well maintained. Altered expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of NAD+-salvage, accompanies the changes in NAD(H) during tumorigenesis. Here, we show by genetic and pharmacological inhibition of NAMPT in glioma cells that fluctuation in intracellular [NAD(H)] differentially affects cell growth and morphodynamics, with motility/invasion capacity showing the highest sensitivity to [NAD(H)] decrease. Extracellular supplementation of NAD+ or re-expression of NAMPT abolished the effects. The effects of NAD(H) decrease on cell motility appeared parallel coupled with diminished pyruvate-lactate conversion by lactate dehydrogenase (LDH) and with changes in intracellular and extracellular pH. The addition of lactic acid rescued and knockdown of LDH-A replicated the effects of [NAD(H)] on motility. Combined, our observations demonstrate that [NAD(H)] is an important metabolic component of cancer cell motility. Nutrient or drug-mediated modulation of NAD(H) levels may therefore represent a new option for blocking the invasive behavior of tumors.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  1. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144(5):646–674. doi:10.1016/j.cell.2011.02.013

    Article  PubMed  CAS  Google Scholar 

  2. Friedl P, Alexander S (2011) Cancer invasion and the microenvironment: plasticity and reciprocity. Cell 147(5):992–1009

    Article  PubMed  CAS  Google Scholar 

  3. Vander Heiden MG, Cantley LC, Thompson CB (2009) Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324(5930):1029–1033

    Article  PubMed  CAS  Google Scholar 

  4. Warburg O (1956) On the origin of cancer cells. Science 123(3191):309–314

    Article  PubMed  CAS  Google Scholar 

  5. Deberardinis RJ, Sayed N, Ditsworth D, Thompson CB (2008) Brick by brick: metabolism and tumor cell growth. Curr Opin Genet Dev 18(1):54–61. doi:10.1016/j.gde.2008.02.003

    Article  PubMed  CAS  Google Scholar 

  6. de Figueiredo LF, Gossmann TI, Ziegler M, Schuster S (2011) Pathway analysis of NAD+ metabolism. Biochem J 439(2):341–348

    Article  PubMed  Google Scholar 

  7. Koch-Nolte F, Fischer S, Haag F, Ziegler M (2011) Compartmentation of NAD+-dependent signalling. FEBS Lett 585(11):1651–1656. doi:10.1016/j.febslet.2011.03.045

    Article  PubMed  CAS  Google Scholar 

  8. de Groof AJ, Te Lindert MM, van Dommelen MM, Wu M, Willemse M, Smift AL, Winer M, Oerlemans F, Pluk H, Fransen JA, Wieringa B (2009) Increased OXPHOS activity precedes rise in glycolytic rate in H-RasV12/E1A transformed fibroblasts that develop a Warburg phenotype. Mol Cancer 8(1):54

    Article  PubMed  Google Scholar 

  9. Garten A, Petzold S, Korner A, Imai S, Kiess W (2009) Nampt: linking NAD biology, metabolism and cancer. Trends Endocrinol Metab 20(3):130–138

    Article  PubMed  CAS  Google Scholar 

  10. Nikiforov A, Dolle C, Niere M, Ziegler M (2011) Pathways and subcellular compartmentation of NAD biosynthesis in human cells: from entry of extracellular precursors to mitochondrial NAD generation. J Biol Chem 286(24):21767–21778. doi:10.1074/jbc.M110.213298

    Article  PubMed  CAS  Google Scholar 

  11. Houtkooper RH, Pirinen E, Auwerx J (2012) Sirtuins as regulators of metabolism and healthspan. Nat Rev Mol Cell Biol 13(4):225–238. doi:10.1038/nrm3293

    PubMed  CAS  Google Scholar 

  12. Bowlby SC, Thomas MJ, D’Agostino RB Jr, Kridel SJ (2012) Nicotinamide phosphoribosyl transferase (Nampt) is required for de novo lipogenesis in tumor cells. PLoS ONE 7(6):e40195. doi:10.1371/journal.pone.0040195

    Article  PubMed  CAS  Google Scholar 

  13. Holen K, Saltz LB, Hollywood E, Burk K, Hanauske AR (2008) The pharmacokinetics, toxicities, and biologic effects of FK866, a nicotinamide adenine dinucleotide biosynthesis inhibitor. Investig New Drugs 26(1):45–51

    Article  CAS  Google Scholar 

  14. Cerna D, Li H, Flaherty S, Takebe N, Coleman CN, Yoo SS (2012) Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) activity by small molecule GMX1778 regulates reactive oxygen species (ROS)-mediated cytotoxicity in a p53- and nicotinic acid phosphoribosyltransferase1 (NAPRT1)-dependent manner. J Biol Chem 287(26):22408–22417. doi:10.1074/jbc.M112.357301

    Article  PubMed  CAS  Google Scholar 

  15. Zhao Y, Jin J, Hu Q, Zhou HM, Yi J, Yu Z, Xu L, Wang X, Yang Y, Loscalzo J (2011) Genetically encoded fluorescent sensors for intracellular NADH detection. Cell Metab 14(4):555–566. doi:10.1016/j.cmet.2011.09.004

    Article  PubMed  CAS  Google Scholar 

  16. Van Horssen R, Galjart N, Rens JA, Eggermont AM, ten Hagen TL (2006) Differential effects of matrix and growth factors on endothelial and fibroblast motility: application of a modified cell migration assay. J Cell Biochem 99(6):1536–1552

    Article  PubMed  Google Scholar 

  17. Murphy DA, Courtneidge SA (2011) The ‘ins’ and ‘outs’ of podosomes and invadopodia: characteristics, formation and function. Nat Rev Mol Cell Biol 12(7):413–426. doi:10.1038/nrm3141

    Article  PubMed  CAS  Google Scholar 

  18. Fantin VR, St-Pierre J, Leder P (2006) Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance. Cancer Cell 9(6):425–434. doi:10.1016/j.ccr.2006.04.023

    Article  PubMed  CAS  Google Scholar 

  19. Le A, Cooper CR, Gouw AM, Dinavahi R, Maitra A, Deck LM, Royer RE, Vander Jagt DL, Semenza GL, Dang CV (2010) Inhibition of lactate dehydrogenase A induces oxidative stress and inhibits tumor progression. Proc Natl Acad Sci USA 107(5):2037–2042. doi:10.1073/pnas.0914433107

    Article  PubMed  CAS  Google Scholar 

  20. Tennant DA, Duran RV, Gottlieb E (2010) Targeting metabolic transformation for cancer therapy. Nat Rev Cancer 10(4):267–277

    Article  PubMed  CAS  Google Scholar 

  21. Gianni D, Diaz B, Taulet N, Fowler B, Courtneidge SA, Bokoch GM (2009) Novel p47(phox)-related organizers regulate localized NADPH oxidase 1 (Nox1) activity. Sci Signal 2(88):ra54. doi:10.1126/scisignal.2000370

  22. Hung RJ, Pak CW, Terman JR (2011) Direct redox regulation of F-actin assembly and disassembly by Mical. Science 334(6063):1710–1713. doi:10.1126/science.1211956

    Article  PubMed  CAS  Google Scholar 

  23. Zhang Q, Wang SY, Nottke AC, Rocheleau JV, Piston DW, Goodman RH (2006) Redox sensor CtBP mediates hypoxia-induced tumor cell migration. Proc Natl Acad Sci USA 103(24):9029–9033. doi:10.1073/pnas.0603269103

    Article  PubMed  CAS  Google Scholar 

  24. Terashima M, Shimoyama M, Tsuchiya M (1999) Introduction of NAD decreases fMLP-induced actin polymerization in chicken polymorphonuclear leukocytes—the role of intracellular ADP-ribosylation of actin for cytoskeletal organization. Biochem Mol Biol Int 47(4):615–620

    PubMed  CAS  Google Scholar 

  25. North BJ, Marshall BL, Borra MT, Denu JM, Verdin E (2003) The human Sir2 ortholog, SIRT2, is an NAD+-dependent tubulin deacetylase. Mol Cell 11(2):437–444

    Article  PubMed  CAS  Google Scholar 

  26. Zhang Y, Zhang M, Dong H, Yong S, Li X, Olashaw N, Kruk PA, Cheng JQ, Bai W, Chen J, Nicosia SV, Zhang X (2009) Deacetylation of cortactin by SIRT1 promotes cell migration. Oncogene 28(3):445–460

    Article  PubMed  CAS  Google Scholar 

  27. Goody MF, Kelly MW, Lessard KN, Khalil A, Henry CA (2010) Nrk2b-mediated NAD+ production regulates cell adhesion and is required for muscle morphogenesis in vivo: Nrk2b and NAD+ in muscle morphogenesis. Dev Biol 344(2):809–826. doi:10.1016/j.ydbio.2010.05.513

    Article  PubMed  CAS  Google Scholar 

  28. Quistorff B, Grunnet N (2011) The isoenzyme pattern of LDH does not play a physiological role; except perhaps during fast transitions in energy metabolism. Aging (Albany NY) 3(5):457–460

    CAS  Google Scholar 

  29. Attanasio F, Caldieri G, Giacchetti G, van Horssen R, Wieringa B, Buccione R (2011) Novel invadopodia components revealed by differential proteomic analysis. Eur J Cell Biol 90(2–3):115–127. doi:10.1016/j.ejcb.2010.05.004

    Article  PubMed  CAS  Google Scholar 

  30. Hirschhaeuser F, Sattler UG, Mueller-Klieser W (2011) Lactate: a metabolic key player in cancer. Cancer Res 71(22):6921–6925

    Article  PubMed  CAS  Google Scholar 

  31. Goetze K, Walenta S, Ksiazkiewicz M, Kunz-Schughart LA, Mueller-Klieser W (2011) Lactate enhances motility of tumor cells and inhibits monocyte migration and cytokine release. Int J Oncol 39(2):453–463. doi:10.3892/ijo.2011.1055

    PubMed  CAS  Google Scholar 

  32. Colen CB, Shen Y, Ghoddoussi F, Yu P, Francis TB, Koch BJ, Monterey MD, Galloway MP, Sloan AE, Mathupala SP (2011) Metabolic targeting of lactate efflux by malignant glioma inhibits invasiveness and induces necrosis: an in vivo study. Neoplasia 13(7):620–632

    PubMed  CAS  Google Scholar 

  33. Bonuccelli G, Tsirigos A, Whitaker-Menezes D, Pavlides S, Pestell RG, Chiavarina B, Frank PG, Flomenberg N, Howell A, Martinez-Outschoorn UE, Sotgia F, Lisanti MP (2010) Ketones and lactate “fuel” tumor growth and metastasis: evidence that epithelial cancer cells use oxidative mitochondrial metabolism. Cell Cycle 9(17):3506–3514

    Article  PubMed  CAS  Google Scholar 

  34. Stock C, Schwab A (2009) Protons make tumor cells move like clockwork. Pflugers Arch 458(5):981–992. doi:10.1007/s00424-009-0677-8

    Article  PubMed  CAS  Google Scholar 

  35. Frantz C, Barreiro G, Dominguez L, Chen X, Eddy R, Condeelis J, Kelly MJ, Jacobson MP, Barber DL (2008) Cofilin is a pH sensor for actin free barbed end formation: role of phosphoinositide binding. J Cell Biol 183(5):865–879. doi:10.1083/jcb.200804161

    Article  PubMed  CAS  Google Scholar 

  36. Magalhaes MA, Larson DR, Mader CC, Bravo-Cordero JJ, Gil-Henn H, Oser M, Chen X, Koleske AJ, Condeelis J (2011) Cortactin phosphorylation regulates cell invasion through a pH-dependent pathway. J Cell Biol 195(5):903–920. doi:10.1083/jcb.201103045

    Article  PubMed  CAS  Google Scholar 

  37. Busco G, Cardone RA, Greco MR, Bellizzi A, Colella M, Antelmi E, Mancini MT, Dell’Aquila ME, Casavola V, Paradiso A, Reshkin SJ (2010) NHE1 promotes invadopodial ECM proteolysis through acidification of the peri-invadopodial space. Faseb J 24(10):3903–3915. doi:10.1096/fj.09-149518

    Article  PubMed  CAS  Google Scholar 

  38. Olson MF, Sahai E (2009) The actin cytoskeleton in cancer cell motility. Clin Exp Metastasis 26(4):273–287

    Article  PubMed  Google Scholar 

  39. Van Horssen R, Janssen E, Peters W, van de Pasch L, Te Lindert MM, van Dommelen MM, Linssen PC, ten Hagen TL, Fransen JA, Wieringa B (2009) Modulation of cell motility by spatial repositioning of enzymatic ATP/ADP exchange capacity. J Biol Chem 284(3):1620–1627

    Article  PubMed  Google Scholar 

  40. Hung YP, Albeck JG, Tantama M, Yellen G (2011) Imaging cytosolic NADH-NAD(+) redox state with a genetically encoded fluorescent biosensor. Cell Metab 14(4):545–554. doi:10.1016/j.cmet.2011.08.012

    Article  PubMed  CAS  Google Scholar 

  41. Koivusalo M, Welch C, Hayashi H, Scott CC, Kim M, Alexander T, Touret N, Hahn KM, Grinstein S (2010) Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling. J Cell Biol 188(4):547–563. doi:10.1083/jcb.200908086

    Article  PubMed  CAS  Google Scholar 

  42. Chiarugi A, Dolle C, Felici R, Ziegler M (2012) The NAD metabolome—a key determinant of cancer cell biology. Nat Rev Cancer 12(11):741–752. doi:10.1038/nrc3340

    Article  PubMed  CAS  Google Scholar 

  43. Yin H, Veer EV, Frontini MJ, Thibert V, O’Neil C, Watson A, Szasz P, Chu MW, Pickering JG (2012) Intrinsic directionality of migrating vascular smooth muscle cells is regulated by NAD+ biosynthesis. J Cell Sci. doi:10.1242/jcs.110262

    Google Scholar 

  44. Zhou M, Zhao Y, Ding Y, Liu H, Liu Z, Fodstad O, Riker AI, Kamarajugadda S, Lu J, Owen LB, Ledoux SP, Tan M (2010) Warburg effect in chemosensitivity: targeting lactate dehydrogenase-A re-sensitizes taxol-resistant cancer cells to taxol. Mol Cancer 9:33. doi:10.1186/1476-4598-9-33

    Article  PubMed  Google Scholar 

  45. Goellner EM, Grimme B, Brown AR, Lin YC, Wang XH, Sugrue KF, Mitchell L, Trivedi RN, Tang JB, Sobol RW (2011) Overcoming temozolomide resistance in glioblastoma via dual inhibition of NAD+ biosynthesis and base excision repair. Cancer Res 71(6):2308–2317. doi:10.1158/0008-5472.can-10-3213

    Article  PubMed  CAS  Google Scholar 

  46. Ho VW, Leung K, Hsu A, Luk B, Lai J, Shen SY, Minchinton AI, Waterhouse D, Bally MB, Lin W, Nelson BH, Sly LM, Krystal G (2011) A low carbohydrate, high protein diet slows tumor growth and prevents cancer initiation. Cancer Res 71(13):4484–4493. doi:10.1158/0008-5472.can-10-3973

    Article  PubMed  CAS  Google Scholar 

  47. Menssen A, Hydbring P, Kapelle K, Vervoorts J, Diebold J, Luscher B, Larsson LG, Hermeking H (2012) The c-MYC oncoprotein, the NAMPT enzyme, the SIRT1-inhibitor DBC1, and the SIRT1 deacetylase form a positive feedback loop. Proc Natl Acad Sci USA 109(4):E187–E196. doi:10.1073/pnas.1105304109

    Article  PubMed  CAS  Google Scholar 

  48. Wang B, Hasan MK, Alvarado E, Yuan H, Wu H, Chen WY (2011) NAMPT overexpression in prostate cancer and its contribution to tumor cell survival and stress response. Oncogene 30(8):907–921. doi:10.1038/onc.2010.468

    Article  PubMed  CAS  Google Scholar 

  49. Wosikowski K, Mattern K, Schemainda I, Hasmann M, Rattel B, Loser R (2002) WK175, a novel antitumor agent, decreases the intracellular nicotinamide adenine dinucleotide concentration and induces the apoptotic cascade in human leukemia cells. Cancer Res 62(4):1057–1062

    PubMed  CAS  Google Scholar 

  50. Dordick RS, Brierley GP, Garlid KD (1980) On the mechanism of A23187-induced potassium efflux in rat liver mitochondria. J Biol Chem 255(21):10299–10305

    PubMed  CAS  Google Scholar 

  51. Krahling H, Mally S, Eble JA, Noel J, Schwab A, Stock C (2009) The glycocalyx maintains a cell surface pH nanoenvironment crucial for integrin-mediated migration of human melanoma cells. Pflugers Arch 458(6):1069–1083. doi:10.1007/s00424-009-0694-7

    Article  PubMed  Google Scholar 

  52. Stock C, Mueller M, Kraehling H, Mally S, Noel J, Eder C, Schwab A (2007) pH nanoenvironment at the surface of single melanoma cells. Cell Physiol Biochem 20(5):679–686

    Article  PubMed  CAS  Google Scholar 

  53. Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S, Boyd MR (1990) New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 82(13):1107 –1112

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Dr. Joost van Schalkwijk and Dr. William Leenders for providing glioma cell lines, Dr. Frank van Leeuwen for NAMPT cDNA, and Dr. Wiljan Hendriks and Dr. Ad de Groof for helpful discussions. This study was supported by Grant KUN-2005-3333 from the Dutch Cancer Society (to BW), Grant Sto 654/3-2 from the Deutsche Forschungsgemeinschaft (to CS and AS), an EMBO Short Term Fellowship and a Vanderes Foundation Research Grant (number 225) (to RvH) and by grants from the Italian Association for Cancer Research (AIRC) and the European Union’s Seventh Framework Program (Grant No. 237946) (to RB).

Author information

Authors and Affiliations

  1. Department of Cell Biology, Nijmegen Centre for Molecular Life Sciences (NCMLS), Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB, Nijmegen, The Netherlands

    Remco van Horssen, Marieke Willemse, Anna Haeger, Tuba Güneri, Jack A. M. Fransen & Bé Wieringa

  2. Tumor Cell Invasion Laboratory, Consorzio Mario Negri Sud, 66030, S. Maria Imbaro (Chieti), Italy

    Francesca Attanasio & Roberto Buccione

  3. Institute of Physiology II, University of Münster, Robert-Koch-Str. 27b, 48149, Münster, Germany

    Albrecht Schwab & Christian M. Stock

Authors
  1. Remco van Horssen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marieke Willemse
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anna Haeger
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Francesca Attanasio
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tuba Güneri
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Albrecht Schwab
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Christian M. Stock
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Roberto Buccione
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jack A. M. Fransen
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Bé Wieringa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bé Wieringa.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 480 kb)

Supplementary Movie 1 (MPG 5738 kb)

Supplementary Movie 2 (MPG 7038 kb)

Supplementary Movie 3 (MPG 7096 kb)

Supplementary Movie 4 (MPG 4316 kb)

Supplementary Movie 5 (MPG 6755 kb)

Supplementary Movie 6 (MPG 6710 kb)

Supplementary Movie 7 (MPG 4261 kb)

Supplementary Movie 8 (MPG 4327 kb)

Supplementary Movie 9 (MPG 4320 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

van Horssen, R., Willemse, M., Haeger, A. et al. Intracellular NAD(H) levels control motility and invasion of glioma cells. Cell. Mol. Life Sci. 70, 2175–2190 (2013). https://doi.org/10.1007/s00018-012-1249-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00018-012-1249-1

Keywords

Search

Navigation