Ukr.Biochem.J. 2016; Volume 88, Issue 2, Mar-Apr, pp. 25-34


Inhibition of IRE1 modifies the hypoxic regulation of GADD family gene expressions in U87 glioma cells

O. H. Minchenko1, I. V. Kryvdiuk1, О. O. Riabovol1,
D. O. Minchenko1,2, S. V. Danilovskyi1, О. O. Ratushna1

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
2Bohomolets National Medical University, Kyiv, Ukraine

We have studied hypoxic regulation of the expression of genes encoded GADD (growth arrest and DNA damage) family proteins in U87 glioma cells in relation to inhibition of IRE1 (inositol requiring enzyme-1), which controls cell proliferation and tumor growth as a central mediator of endoplasmic reticulum stress. We have shown that hypoxia up-regulates the expression of GADD34, GADD45A, GADD45B, and GADD153 genes, which are related to cell proliferation and apoptosis, in control (transfected by empty vector) glioma cells in gene specific manner. At the same time, the expression level of EIF2AK1 (eukaryotic translation initiation factor 2-alpha kinase 1) and AIFM1 (apoptosis inducing factor, mitochondria associated 1) genes in these cells is down-regulated upon hypoxic condition. It was also shown that inhibition of ІRE1 signaling enzyme function in U87 glioma cells enhances the effect of hypoxia on these genes expression, except EIF2AK1 and AIFM1 genes. Furthermore, the expression of all studied genes in ІRE1 knockdown cells is significantly decreased upon normoxic condition, except GADD45B gene, which expression level is strongly up-regulated. Therefore, the expression level of genes encoding GADD34, GADD45A, GADD45B, GADD153, EIF2AK1, and AIFM1 is affected by hypoxia and by inhibition of IRE1-mediated endoplasmic reticulum stress signaling in gene specific manner and correlates with suppression of glioma cell proliferation upon inhibition of the IRE1 enzyme function.

Keywords: , , , , , , , , ,


  1. Moenner M, Pluquet O, Bouchecareilh M, Chevet E. Integrated endoplasmic reticulum stress responses in cancer. Cancer Res. 2007 Nov 15;67(22):10631-4. PubMed, CrossRef
  2. Schröder M. Endoplasmic reticulum stress responses. Cell Mol Life Sci. 2008 Mar;65(6):862-94. Review. PubMed, CrossRef
  3. Wang S, Kaufman RJ. The impact of the unfolded protein response on human disease. J Cell Biol. 2012 Jun 25;197(7):857-67. Review. PubMed, PubMedCentral, CrossRef
  4. Auf G, Jabouille A, Guérit S, Pineau R, Delugin M, Bouchecareilh M, Magnin N, Favereaux A, Maitre M, Gaiser T, von Deimling A, Czabanka M, Vajkoczy P, Chevet E, Bikfalvi A, Moenner M. Inositol-requiring enzyme 1alpha is a key regulator of angiogenesis and invasion in malignant glioma. Proc Natl Acad Sci USA. 2010 Aug 31;107(35):15553-8.  PubMed, PubMedCentral, CrossRef
  5. Drogat B, Auguste P, Nguyen DT, Bouchecareilh M, Pineau R, Nalbantoglu J, Kaufman RJ, Chevet E, Bikfalvi A, Moenner M. IRE1 signaling is essential for ischemia-induced vascular endothelial growth factor-A expression and contributes to angiogenesis and tumor growth in vivo. Cancer Res. 2007 Jul 15;67(14):6700-7. PubMed
  6. Lenihan CR, Taylor CT. The impact of hypoxia on cell death pathways. Biochem Soc Trans. 2013 Apr;41(2):657-63. Review. PubMed, CrossRef
  7. Denko NC. Hypoxia, HIF1 and glucose metabolism in the solid tumour. Nat Rev Cancer. 2008 Sep;8(9):705-13. PubMed, CrossRef
  8. Hetz C, Chevet E, Harding HP. Targeting the unfolded protein response in disease. Nat Rev Drug Discov. 2013 Sep;12(9):703-19. Review. PubMed, CrossRef
  9. Manié SN, Lebeau J, Chevet E. Cellular mechanisms of endoplasmic reticulum stress signaling in health and disease. 3. Orchestrating the unfolded protein response in oncogenesis: an update. Am J Physiol Cell Physiol. 2014 Nov 15;307(10):C901-7. Review. PubMed, CrossRef
  10. Hollien J, Lin JH, Li H, Stevens N, Walter P, Weissman JS. Regulated Ire1-dependent decay of messenger RNAs in mammalian cells. J Cell Biol. 2009 Aug 10;186(3):323-31. PubMed, PubMedCentral, CrossRef
  11. Tampakakis E, Tabit CE, Holbrook M, Linder EA, Berk BD, Frame AA, Bretón-Romero R, Fetterman JL, Gokce N, Vita JA, Hamburg NM. Intravenous Lipid Infusion Induces Endoplasmic Reticulum Stress in Endothelial Cells and Blood Mononuclear Cells of Healthy Adults. J Am Heart Assoc. 2016 Jan 11;5(1). pii: e002574. PubMed, CrossRef
  12. Choy MS, Yusoff P, Lee IC, Newton JC, Goh CW, Page R, Shenolikar S, Peti W. Structural and Functional Analysis of the GADD34:PP1 eIF2α Phosphatase. Cell Rep. 2015 Jun 30;11(12):1885-91. PubMed, PubMedCentral, CrossRef
  13. Young SK, Willy JA, Wu C, Sachs MS, Wek RC. Ribosome Reinitiation Directs Gene-specific Translation and Regulates the Integrated Stress Response. J Biol Chem. 2015 Nov 20;290(47):28257-71. PubMed, PubMedCentral, CrossRef
  14. Lee SK, Kim YS. Phosphorylation of eIF2α attenuates statin-induced apoptosis by inhibiting the stabilization and translocation of p53 to the mitochondria. Int J Oncol. 2013 Mar;42(3):810-6. PubMed, PubMedCentral, CrossRef
  15. Liu L, Ito S, Nishio N, Sun Y, Chen N, Tanaka Y, Isobe K. GADD34 Facilitates Cell Death Resulting from Proteasome Inhibition. Anticancer Res. 2015 Oct;35(10):5317-24. PubMed
  16. Hoffman B, Liebermann DA. Gadd45 in modulation of solid tumors and leukemia. Adv Exp Med Biol. 2013;793:21-33. Review. PubMed, CrossRef
  17. Salvador JM, Brown-Clay JD, Fornace AJ Jr. Gadd45 in stress signaling, cell cycle control, and apoptosis. Adv Exp Med Biol. 2013;793:1-19. Review. PubMed, CrossRef
  18. Zhang XY; Xun-Qu, Wang CQ, Liu GX, Zhou CJ, Wang ZG. Expression of growth arrest and DNA damage inducible 45a in human oral squamous cell carcinoma is associated with tumor progression and clinical outcome. J Cancer Res Ther. 2014 Nov;10 Suppl:C108-13. PubMed, CrossRef
  19. Michaelis KA, Knox AJ, Xu M, Kiseljak-Vassiliades K, Edwards MG, Geraci M, Kleinschmidt-DeMasters BK, Lillehei KO, Wierman ME. Identification of growth arrest and DNA-damage-inducible gene beta (GADD45beta) as a novel tumor suppressor in pituitary gonadotrope tumors. Endocrinology. 2011 Oct;152(10):3603-13. PubMed, PubMedCentral, CrossRef
  20. Bifulco G, Miele C, Di Jeso B, Beguinot F, Nappi C, Di Carlo C, Capuozzo S, Terrazzano G, Insabato L, Ulianich L. Endoplasmic reticulum stress is activated in endometrial adenocarcinoma. Gynecol Oncol. 2012 Apr;125(1):220-5. PubMed, CrossRef
  21. Li T, Su L, Lei Y, Liu X, Zhang Y, Liu X. DDIT3 and KAT2A Proteins Regulate TNFRSF10A and TNFRSF10B Expression in Endoplasmic Reticulum Stress-mediated Apoptosis in Human Lung Cancer Cells. J Biol Chem. 2015 Apr 24;290(17):11108-18. PubMed, PubMedCentral, CrossRef
  22. Tian X, Ye J, Alonso-Basanta M, Hahn SM, Koumenis C, Dorsey JF. Modulation of CCAAT/enhancer binding protein homologous protein (CHOP)-dependent DR5 expression by nelfinavir sensitizes glioblastoma multiforme cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). J Biol Chem. 2011 Aug 19;286(33):29408-16. PubMed, PubMedCentral, CrossRef
  23. Palam LR, Baird TD, Wek RC. Phosphorylation of eIF2 facilitates ribosomal bypass of an inhibitory upstream ORF to enhance CHOP translation. J Biol Chem. 2011 Apr 1;286(13):10939-49. PubMed, PubMedCentral, CrossRef
  24. Haapa-Paananen S, Chen P, Hellström K, Kohonen P, Hautaniemi S, Kallioniemi O, Perälä M. Functional profiling of precursor MicroRNAs identifies MicroRNAs essential for glioma proliferation. PLoS One. 2013;8(4):e60930. PubMed, PubMedCentral, CrossRef
  25. Chatterjee S, Panda AC, Berwal SK, Sreejith RK, Ritvika C, Seshadri V, Pal JK. Vimentin is a component of a complex that binds to the 5′-UTR of human heme-regulated eIF2α kinase mRNA and regulates its translation. FEBS Lett. 2013 Mar 1;587(5):474-80. PubMed, CrossRef
  26. Cobanoglu B, Ceyran AB, Simsek M, Şenol S. Immunohistochemical analysis of Bax and AIF in colorectal tumors. Int J Clin Exp Med. 2015 Sep 15;8(9):16071-6. eCollection 2015. PubMed, PubMedCentral
  27. Zhao H, Wang C, Lu B, Zhou Z, Jin Y, Wang Z, Zheng L, Liu K, Luo T, Zhu D, Chi G, Luo Y, Ge P. Pristimerin triggers AIF-dependent programmed necrosis in glioma cells via activation of JNK. Cancer Lett. 2016 Apr 28;374(1):136-48. PubMed, CrossRef
  28. Minchenko DO, Danilovskyi SV, Kryvdiuk IV, Bakalets TV, Lypova NM, Karbovsky LL, Minchenko OH. Inhibition of ERN1 modifies the hypoxic regulation of the expression of TP53-related genes in U87 glioma cells. Endoplasm Reticul Stress Dis. 2014; 1(1): 18-26. CrossRef
  29. Minchenko O, Opentanova I, Minchenko D, Ogura T, Esumi H. Hypoxia induces transcription of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-4 gene via hypoxia-inducible factor-1alpha activation. FEBS Lett. 2004 Oct 8;576(1-2):14-20.  PubMed, CrossRef
  30. Bochkov VN, Philippova M, Oskolkova O, Kadl A, Furnkranz A, Karabeg E, Afonyushkin T, Gruber F, Breuss J, Minchenko A, Mechtcheriakova D, Hohensinner P, Rychli K, Wojta J, Resink T, Erne P, Binder BR, Leitinger N. xidized phospholipids stimulate angiogenesis via autocrine mechanisms, implicating a novel role for lipid oxidation in the evolution of atherosclerotic lesions. Circ Res. 2006 Oct 13;99(8):900-8. PubMed, CrossRef
  31. Minchenko DО, Karbovskyi LL, Danilovskyi SV, Moenner M, Minchenko OH. Effect of hypoxia and glutamine or glucose deprivation on the expression of retinoblastoma and retinoblastoma-related genes in ERN1 knockdown glioma U87 cell line. Am J Mol Biol. 2012; 2(1): 21-31. CrossRef
  32. Minchenko DO, Kharkova AP, Karbovskyi LL, Minchenko OH. Expression of insulin-like growth factor binding protein genes and its hypoxic regulation in U87 glioma cells depends on ERN1 mediated signaling pathway of endoplasmic reticulum stress. Endocr Regul. 2015 Apr;49(2):73-83. PubMed, CrossRef
  33.  Minchenko OH, Kharkova AP, Minchenko DO, Karbovskyi LL. Effect of hypoxia on the expression of IGFBP6, IGFBP7, NOV, CYR61, WISP1, and WISP2 genes in U87 glioma cells depends on ERN1 mediated signaling pathway of endoplasmic reticulum stress. Ukr Biochem J. 2015; 87(6): 52-63. CrossRef
  34. Koumenis C. ER stress, hypoxia tolerance and tumor progression. Curr Mol Med. 2006 Feb;6(1):55-69. Review. PubMed, CrossRef
  35. Bi M, Naczki C, Koritzinsky M, Fels D, Blais J, Hu N, Harding H, Novoa I, Varia M, Raleigh J, Scheuner D, Kaufman RJ, Bell J, Ron D, Wouters BG, Koumenis C. ER stress-regulated translation increases tolerance to extreme hypoxia and promotes tumor growth. EMBO J. 2005 Oct 5;24(19):3470-81. PubMed, PubMedCentral, CrossRef
  36. Koumenis C, Naczki C, Koritzinsky M, Rastani S, Diehl A, Sonenberg N, Koromilas A, Wouters BG. Regulation of protein synthesis by hypoxia via activation of the endoplasmic reticulum kinase PERK and phosphorylation of the translation initiation factor eIF2alpha. Mol Cell Biol. 2002 Nov;22(21):7405-16. PubMed, PubMedCentral, CrossRef
  37. Auf G, Jabouille A, Delugin M, Guérit S, Pineau R, North S, Platonova N, Maitre M, Favereaux A, Vajkoczy P, Seno M, Bikfalvi A, Minchenko D, Minchenko O, Moenner M. High epiregulin expression in human U87 glioma cells relies on IRE1α and promotes autocrine growth through EGF receptor. BMC Cancer. 2013 Dec 13;13:597. PubMed, PubMedCentral, CrossRef

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