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Knockdown of GluR1 expression by RNA interference inhibits glioma proliferation

  • lab. investigation-human/animal tissue
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Abstract

High-grade gliomas release excitotoxic concentrations of glutamate which contributes to their malignant phenotype. To improve our understanding of the mechanisms by which glutamate enhances tumor growth and invasion, we examined α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-mediated signaling in glioma cell lines. shRNA was used to stably knockdown GluR1, the most abundant AMPA receptor subunit in glioma, to evaluate its role in tumor signaling, proliferation and tumorigenicity. In a tissue array, there was a statistically significant increase in GluR1 expression in glioblastoma samples compared to anaplastic astrocytoma and low-grade tumors. In vitro, we observed a time and dose-dependent increase in MAPK phosphorylation following exposure to AMPA, which was blocked with AMPA receptor antagonists and the MEK1 inhibitor PD98059. Retroviral delivery of GluR1 shRNA in U251 and U87 glioma cells reduced GluR1 protein expression, inhibited AMPA-mediated increases in MAPK phosphorylation, and decreased glioma proliferation in vitro. U251 and U87 shGluR1 cells implanted into the flanks of nude mice grew slower than controls, which correlated with a decrease in proliferation measured by Ki-67 staining and an increase in apoptosis. These results suggest that AMPA receptors are abundantly expressed in high-grade gliomas and gene silencing of the GluR1 AMPA receptor subunit results in abrogation of AMPA-mediated signaling and tumor growth.

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References

  1. Behrens PF, Langemann H, Strohschein R, Draeger J, Hennig J (2000) Extracellular glutamate and other metabolites in and around RG2 rat glioma: an intracerebral microdialysis study. J Neurooncol 47:11–22

    Article  PubMed  CAS  Google Scholar 

  2. Roslin M, Henriksson R, Bergstrom P, Ungerstedt U, Bergenheim AT (2003) Baseline levels of glucose metabolites, glutamate and glycerol in malignant glioma assessed by stereotactic microdialysis. J Neurooncol 61:151–160

    Article  PubMed  Google Scholar 

  3. Ye ZC, Sontheimer H (1999) Glioma cells release excitotoxic concentrations of glutamate. Cancer Res 59:4383–4391

    PubMed  CAS  Google Scholar 

  4. Chung WJ, Lyons SA, Nelson GM, Hamza H, Gladson CL, Gillespie GY, Sontheimer H (2005) Inhibition of cystine uptake disrupts the growth of primary brain tumors. J Neurosci 25:7101–7110

    Article  PubMed  CAS  Google Scholar 

  5. de Groot JF, Liu TJ, Fuller G, Yung WK (2005) The excitatory amino acid transporter-2 induces apoptosis and decreases glioma growth in vitro and in vivo. Cancer Res 65:1934–1940

    Article  PubMed  Google Scholar 

  6. Ye ZC, Rothstein JD, Sontheimer H (1999) Compromised glutamate transport in human glioma cells: reduction-mislocalization of sodium-dependent glutamate transporters and enhanced activity of cystine-glutamate exchange. J Neurosci 19:10767–10777

    PubMed  CAS  Google Scholar 

  7. Rzeski W, Turski L, Ikonomidou C (2001) Glutamate antagonists limit tumor growth. Proc Natl Acad Sci USA 98:6372–6377

    Article  PubMed  CAS  Google Scholar 

  8. Takano T, Lin JH, Arcuino G, Gao Q, Yang J, Nedergaard M (2001) Glutamate release promotes growth of malignant gliomas. Nat Med 7:1010–1015

    Article  PubMed  CAS  Google Scholar 

  9. D’Onofrio M, Arcella A, Bruno V, Ngomba RT, Battaglia G, Lombari V, Ragona G, Calogero A, Nicoletti F (2003) Pharmacological blockade of mGlu2/3 metabotropic glutamate receptors reduces cell proliferation in cultured human glioma cells. J Neurochem 84:1288–1295

    Article  PubMed  CAS  Google Scholar 

  10. Ishiuchi S, Tsuzuki K, Yoshida Y, Yamada N, Hagimura N, Okado H, Miwa A, Kurihara H, Nakazato Y, Tamura M, Sasaki T, Ozawa S (2002) Blockage of Ca(2+)-permeable AMPA receptors suppresses migration and induces apoptosis in human glioblastoma cells. Nat Med 8:971–978

    Article  PubMed  CAS  Google Scholar 

  11. Rzeski W, Ikonomidou C, Turski L (2002) Glutamate antagonists limit tumor growth. Biochem Pharmacol 64:1195–1200

    Article  PubMed  CAS  Google Scholar 

  12. Dingledine R, Borges K, Bowie D, Traynelis SF (1999) The glutamate receptor ion channels. Pharmacol Rev 51:7–61

    PubMed  CAS  Google Scholar 

  13. Hayashi T, Umemori H, Mishina M, Yamamoto T (1999) The AMPA receptor interacts with and signals through the protein tyrosine kinase Lyn. Nature 397:72–76

    Article  PubMed  CAS  Google Scholar 

  14. Wang Y, Durkin JP (1995) alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, but not N-methyl-d-aspartate, activates mitogen-activated protein kinase through G-protein beta gamma subunits in rat cortical neurons. J Biol Chem 270:22783–22787

    Article  PubMed  CAS  Google Scholar 

  15. Maas S, Patt S, Schrey M, Rich A (2001) Underediting of glutamate receptor GluR-B mRNA in malignant gliomas. Proc Natl Acad Sci USA 98:14687–14692

    Article  PubMed  CAS  Google Scholar 

  16. Ishiuchi S, Yoshida Y, Sugawara K, Aihara M, Ohtani T, Watanabe T, Saito N, Tsuzuki K, Okado H, Miwa A, Nakazato Y, Ozawa S (2007) Ca2+-permeable AMPA receptors regulate growth of human glioblastoma via Akt activation. J Neurosci 27:7987–8001

    Article  PubMed  CAS  Google Scholar 

  17. Schenk U, Menna E, Kim T, Passafaro M, Chang S, De Camilli P, Matteoli M (2005) A novel pathway for presynaptic mitogen-activated kinase activation via AMPA receptors. J Neurosci 25:1654–1663

    Article  PubMed  CAS  Google Scholar 

  18. Reddy KB, Nabha SM, Atanaskova N (2003) Role of MAP kinase in tumor progression and invasion. Cancer Metastasis Rev 22:395–403

    Article  PubMed  CAS  Google Scholar 

  19. Newton HB (2003) Molecular neuro-oncology and development of targeted therapeutic strategies for brain tumors. Part 1: Growth factor and Ras signaling pathways. Expert Rev Anticancer Ther 3:595–614

    Article  PubMed  CAS  Google Scholar 

  20. Rajasekhar VK, Viale A, Socci ND, Wiedmann M, Hu X, Holland EC (2003) Oncogenic Ras and Akt signaling contribute to glioblastoma formation by differential recruitment of existing mRNAs to polysomes. Mol Cell 12:889–901

    Article  PubMed  CAS  Google Scholar 

  21. Hao X, Sun B, Hu L, Lahdesmaki H, Dunmire V, Feng Y, Zhang SW, Wang H, Wu C, Fuller GN, Symmans WF, Shmulevich I, Zhang W (2004) Differential gene and protein expression in primary breast malignancies and their lymph node metastases as revealed by combined cDNA microarray and tissue microarray analysis. Cancer 100:1110–1122

    Article  PubMed  CAS  Google Scholar 

  22. Wang H, Shen W, Huang H, Hu L, Ramdas L, Zhou YH, Liao WS, Fuller GN, Zhang W (2003) Insulin-like growth factor binding protein 2 enhances glioblastoma invasion by activating invasion-enhancing genes. Cancer Res 63:4315–4321

    PubMed  CAS  Google Scholar 

  23. Wang H, Zhang W, Fuller GN (2002) Tissue microarrays: applications in neuropathology research, diagnosis, and education. Brain Pathol 12:95–107

    Article  PubMed  Google Scholar 

  24. Yamada KA, Tang CM (1993) Benzothiadiazides inhibit rapid glutamate receptor desensitization and enhance glutamatergic synaptic currents. J Neurosci 13:3904–3915

    PubMed  CAS  Google Scholar 

  25. Paddison PJ, Cleary M, Silva JM, Chang K, Sheth N, Sachidanandam R, Hannon GJ (2004) Cloning of short hairpin RNAs for gene knockdown in mammalian cells. Nat Methods 1:163–167

    Article  PubMed  CAS  Google Scholar 

  26. Falch E, Brehm L, Mikkelsen I, Johansen TN, Skjaerbaek N, Nielsen B, Stensbol TB, Ebert B, Krogsgaard-Larsen P (1998) Heteroaryl analogues of AMPA. 2. Synthesis, absolute stereochemistry, photochemistry, and structure-activity relationships. J Med Chem 41:2513–2523

    Article  PubMed  CAS  Google Scholar 

  27. Sontheimer H (2003) Malignant gliomas: perverting glutamate and ion homeostasis for selective advantage. Trends Neurosci 26:543–549

    Article  PubMed  CAS  Google Scholar 

  28. Krizbai IA, Deli MA, Pestenacz A, Siklos L, Szabo CA, Andras I, Joo F (1998) Expression of glutamate receptors on cultured cerebral endothelial cells. J Neurosci Res 54:814–819

    Article  PubMed  CAS  Google Scholar 

  29. Morley P, Small DL, Murray CL, Mealing GA, Poulter MO, Durkin JP, Stanimirovic DB (1998) Evidence that functional glutamate receptors are not expressed on rat or human cerebromicrovascular endothelial cells. J Cereb Blood Flow Metab 18:396–406

    Article  PubMed  CAS  Google Scholar 

  30. Andras IE, Deli MA, Veszelka S, Hayashi K, Hennig B, Toborek M (2007) The NMDA and AMPA/KA receptors are involved in glutamate-induced alterations of occludin expression and phosphorylation in brain endothelial cells. J Cereb Blood Flow Metab 8:1431–1443

    Article  CAS  Google Scholar 

  31. Troppmair J, Bruder JT, Munoz H, Lloyd PA, Kyriakis J, Banerjee P, Avruch J, Rapp UR (1994) Mitogen-activated protein kinase/extracellular signal-regulated protein kinase activation by oncogenes, serum, and 12-O-tetradecanoylphorbol-13-acetate requires Raf and is necessary for transformation. J Biol Chem 269:7030–7035

    PubMed  CAS  Google Scholar 

  32. Besson A, Yong VW (2001) Mitogenic signaling and the relationship to cell cycle regulation in astrocytomas. J Neurooncol 51:245–264

    Article  PubMed  CAS  Google Scholar 

  33. Bollag G, McCormick F (1991) Regulators and effectors of ras proteins. Annu Rev Cell Biol 7:601–632

    Article  PubMed  CAS  Google Scholar 

  34. Rosen LB, Ginty DD, Weber MJ, Greenberg ME (1994) Membrane depolarization and calcium influx stimulate MEK and MAP kinase via activation of Ras. Neuron 12:1207–1221

    Article  PubMed  CAS  Google Scholar 

  35. Calautti E, Missero C, Stein PL, Ezzell RM, Dotto GP (1995) fyn tyrosine kinase is involved in keratinocyte differentiation control. Genes Dev 9:2279–2291

    Article  PubMed  CAS  Google Scholar 

  36. Luo JH, Kahn S, O’Driscoll K, Weinstein IB (1993) The regulatory domain of protein kinase C beta 1 contains phosphatidylserine- and phorbol ester-dependent calcium binding activity. J Biol Chem 268:3715–3719

    PubMed  CAS  Google Scholar 

  37. Yano S, Tokumitsu H, Soderling TR (1998) Calcium promotes cell survival through CaM-K kinase activation of the protein-kinase-B pathway. Nature 396:584–587

    Article  PubMed  CAS  Google Scholar 

  38. Czauderna F, Santel A, Hinz M, Fechtner M, Durieux B, Fisch G, Leenders F, Arnold W, Giese K, Klippel A, Kaufmann J (2003) Inducible shRNA expression for application in a prostate cancer mouse model. Nucleic Acids Res 31:e127

    Article  PubMed  CAS  Google Scholar 

  39. Kim E, Sheng M (2004) PDZ domain proteins of synapses. Nat Rev Neurosci 5:771–781

    Article  PubMed  CAS  Google Scholar 

  40. Sheng M (2001) Molecular organization of the postsynaptic specialization. Proc Natl Acad Sci USA 98:7058–7061

    Article  PubMed  CAS  Google Scholar 

  41. Bruckner K, Pablo Labrador J, Scheiffele P, Herb A, Seeburg PH, Klein R (1999) EphrinB ligands recruit GRIP family PDZ adaptor proteins into raft membrane microdomains. Neuron 22:511–524

    Article  PubMed  CAS  Google Scholar 

  42. Ye B, Liao D, Zhang X, Zhang P, Dong H, Huganir RL (2000) GRASP-1: a neuronal RasGEF associated with the AMPA receptor/GRIP complex. Neuron 26:603–617

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

Financial Support provided by an Institutional Research Grant from The University of Texas M. D. Anderson Cancer Center to JF de Groot. We would like to thank Lynda Corley for technical assistance with the tissue microarray and Pierrette Lo for thoughtful editing of this manuscript.

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Authors and Affiliations

  1. Brain Tumor Center, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA

    John F. de Groot, Yuji Piao, Li Lu & W. K. Alfred Yung

  2. Department of Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA

    Gregory N. Fuller

  3. Department of Neuro-Oncology, Unit 431, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX, 77030, USA

    John F. de Groot

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  1. John F. de Groot
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Correspondence to John F. de Groot.

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de Groot, J.F., Piao, Y., Lu, L. et al. Knockdown of GluR1 expression by RNA interference inhibits glioma proliferation. J Neurooncol 88, 121–133 (2008). https://doi.org/10.1007/s11060-008-9552-2

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  • DOI: https://doi.org/10.1007/s11060-008-9552-2

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