Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

HRI-mediated translational repression reduces proteotoxicity and sensitivity to bortezomib in human pancreatic cancer cells

Oncogene volume 37, pages 4413–4427 (2018)Cite this article

Subjects

Abstract

Human cancer cells display extensive heterogeneity in their sensitivities to the proteasome inhibitor bortezomib (Velcade). The molecular mechanisms underlying this heterogeneity remain unclear, and strategies to overcome resistance are limited. Here, we discover that inherent differences in eIF2α phosphorylation among a panel of ten human pancreatic cancer cell lines significantly impacts bortezomib sensitivity, and implicate the HRI (heme-regulated inhibitor) eIF2α kinase as a novel therapeutic target. Within our panel, we identified a subset of cell lines with defective induction of eIF2α phosphorylation, conferring a high degree of sensitivity to bortezomib. These bortezomib-sensitive cells exhibited impaired translation attenuation followed by toxic accumulation of protein aggregates and reactive oxygen species (ROS), whereas the bortezomib-resistant cell lines displayed increased phosphorylation of eIF2α, decreased translation, few protein aggregates, and minimal ROS production. Importantly, we identified HRI as the primary bortezomib-activated eIF2α kinase, and demonstrated that HRI knockdown promoted cell death in the bortezomib-resistant cells. Overall, our data implicate inducible HRI-mediated phosphorylation of eIF2α as a central cytoprotective mechanism following exposure to bortezomib and provide proof-of-concept for the development of HRI inhibitors to overcome proteasome inhibitor resistance.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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

Similar content being viewed by others

References

  1. Adams J, Kauffman M. Development of the proteasome inhibitor Velcade (Bortezomib). Cancer Invest. 2004;22:304–11.

    Article  PubMed  CAS  Google Scholar 

  2. Goldberg AL. Probing the proteasome pathway. Nat Biotechnol. 2000;18:494–6.

    Article  PubMed  CAS  Google Scholar 

  3. Jiang HY, Wek RC. Phosphorylation of the alpha-subunit of the eukaryotic initiation factor-2 (eIF2alpha) reduces protein synthesis and enhances apoptosis in response to proteasome inhibition. J Biol Chem. 2005;280:14189–202.

    Article  PubMed  CAS  Google Scholar 

  4. Yerlikaya A, Kimball SR, Stanley BA. Phosphorylation of eIF2alpha in response to 26S proteasome inhibition is mediated by the haem-regulated inhibitor (HRI) kinase. Biochem J. 2008;412:579–88.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Obeng EA, Carlson LM, Gutman DM, Harrington WJ Jr, Lee KP, Boise LH. Proteasome inhibitors induce a terminal unfolded protein response in multiple myeloma cells. Blood. 2006;107:4907–16.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Lee DH, Goldberg AL. Proteasome inhibitors cause induction of heat shock proteins and trehalose, which together confer thermotolerance in Saccharomyces cerevisiae. Mol Cell Biol. 1998;18:30–8.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Zhu K, Dunner K Jr, McConkey DJ. Proteasome inhibitors activate autophagy as a cytoprotective response in human prostate cancer cells. Oncogene. 2010;29:451–62.

    Article  PubMed  CAS  Google Scholar 

  8. Baird TD, Wek RC. Eukaryotic initiation factor 2 phosphorylation and translational control in metabolism. Adv Nutr. 2012;3:307–21.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Harding HP, Zhang Y, Zeng H, Novoa I, Lu PD, Calfon M, et al. An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Mol Cell. 2003;11:619–33.

    Article  PubMed  CAS  Google Scholar 

  10. Han AP, Yu C, Lu L, Fujiwara Y, Browne C, Chin G, et al. Heme-regulated eIF2alpha kinase (HRI) is required for translational regulation and survival of erythroid precursors in iron deficiency. EMBO J. 2001;20:6909–18.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Zhang F, Romano PR, Nagamura-Inoue T, Tian B, Dever TE, Mathews MB, et al. Binding of double-stranded RNA to protein kinase PKR is required for dimerization and promotes critical autophosphorylation events in the activation loop. J Biol Chem. 2001;276:24946–58.

    Article  PubMed  CAS  Google Scholar 

  12. Walter P, Ron D. The unfolded protein response: from stress pathway to homeostatic regulation. Science. 2011;334:1081–6.

    Article  PubMed  CAS  Google Scholar 

  13. Wek SA, Zhu S, Wek RC. The histidyl-tRNA synthetase-related sequence in the eIF-2 alpha protein kinase GCN2 interacts with tRNA and is required for activation in response to starvation for different amino acids. Mol Cell Biol. 1995;15:4497–506.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Taniuchi S, Miyake M, Tsugawa K, Oyadomari M, Oyadomari S. Integrated stress response of vertebrates is regulated by four eIF2alpha kinases. Sci Rep. 2016;6:32886.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Balch WE, Morimoto RI, Dillin A, Kelly JW. Adapting proteostasis for disease intervention. Science. 2008;319:916–9.

    Article  PubMed  CAS  Google Scholar 

  16. Ross CA, Poirier MA. Protein aggregation and neurodegenerative disease. Nat Med. 2004;10:S10–S7.

    Article  PubMed  CAS  Google Scholar 

  17. Jones RG, Thompson CB. Tumor suppressors and cell metabolism: a recipe for cancer growth. Genes Dev. 2009;23:537–48.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Solimini NL, Luo J, Elledge SJ. Non-oncogene addiction and the stress phenotype of cancer cells. Cell. 2007;130:986–8.

    Article  PubMed  CAS  Google Scholar 

  19. Xu W, Trepel J, Neckers L. Ras, ROS and proteotoxic stress: a delicate balance. Cancer Cell. 2011;20:281–2.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Chauhan D, Hideshima T, Anderson KC. Proteasome inhibition in multiple myeloma: therapeutic implication. Annu Rev Pharmacol Toxicol. 2005;45:465–76.

    Article  PubMed  CAS  Google Scholar 

  21. Kane RC, Dagher R, Farrell A, Ko CW, Sridhara R, Justice R, et al. Bortezomib for the treatment of mantle cell lymphoma. Clin Cancer Res. 2007;13(18 Pt 1):5291–4.

    Article  PubMed  CAS  Google Scholar 

  22. Nawrocki ST, Carew JS, Pino MS, Highshaw RA, Andtbacka RH, Dunner K Jr, et al. Aggresome disruption: a novel strategy to enhance bortezomib-induced apoptosis in pancreatic cancer cells. Cancer Res. 2006;66:3773–81.

    Article  PubMed  CAS  Google Scholar 

  23. Zhu K, Chan W, Heymach J, Wilkinson M, McConkey DJ. Control of HIF-1alpha expression by eIF2 alpha phosphorylation-mediated translational repression. Cancer Res. 2009;69:1836–43.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Knutsen JH, Rodland GE, Boe CA, Haland TW, Sunnerhagen P, Grallert B, et al. Stress-induced inhibition of translation independently of eIF2alpha phosphorylation. J Cell Sci. 2015;128:4420–7.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Leu JI, Pimkina J, Frank A, Murphy ME, George DL. A small molecule inhibitor of inducible heat shock protein 70. Mol Cell. 2009;36:15–27.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Wyttenbach A, Sauvageot O, Carmichael J, Diaz-Latoud C, Arrigo AP, Rubinsztein DC. Heat shock protein 27 prevents cellular polyglutamine toxicity and suppresses the increase of reactive oxygen species caused by huntingtin. Hum Mol Genet. 2002;11:1137–51.

    Article  PubMed  CAS  Google Scholar 

  27. Tabner BJ, Turnbull S, El-Agnaf OM, Allsop D. Formation of hydrogen peroxide and hydroxyl radicals from A(beta) and alpha-synuclein as a possible mechanism of cell death in Alzheimer’s disease and Parkinson’s disease. Free Radic Biol Med. 2002;32:1076–83.

    Article  PubMed  CAS  Google Scholar 

  28. Bucciantini M, Calloni G, Chiti F, Formigli L, Nosi D, Dobson CM, et al. Prefibrillar amyloid protein aggregates share common features of cytotoxicity. J Biol Chem. 2004;279:31374–82.

    Article  PubMed  CAS  Google Scholar 

  29. Jenner P. Oxidative stress in Parkinson’s disease. Ann Neurol. 2003;53 Suppl 3:S26–36. discussion S-8.

    Article  PubMed  CAS  Google Scholar 

  30. Wu D, Yotnda P. Production and detection of reactive oxygen species (ROS) in cancers. J Vis Exp. 2011; pii:3357; https://doi.org/10.3791/3357.

  31. Chen S, Blank JL, Peters T, Liu XJ, Rappoli DM, Pickard MD, et al. Genome-wide siRNA screen for modulators of cell death induced by proteasome inhibitor bortezomib. Cancer Res. 2010;70:4318–26.

    Article  PubMed  CAS  Google Scholar 

  32. Meister S, Schubert U, Neubert K, Herrmann K, Burger R, Gramatzki M, et al. Extensive immunoglobulin production sensitizes myeloma cells for proteasome inhibition. Cancer Res. 2007;67:1783–92.

    Article  PubMed  CAS  Google Scholar 

  33. Schewe DM, Aguirre-Ghiso JA. Inhibition of eIF2alpha dephosphorylation maximizes bortezomib efficiency and eliminates quiescent multiple myeloma cells surviving proteasome inhibitor therapy. Cancer Res. 2009;69:1545–52.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Vilas-Boas Fde A, da Silva AM, de Sousa LP, Lima KM, Vago JP, Bittencourt LF, et al. Impairment of stress granule assembly via inhibition of the eIF2alpha phosphorylation sensitizes glioma cells to chemotherapeutic agents. J Neurooncol. 2016;127:253–60.

    Article  PubMed  CAS  Google Scholar 

  35. Miller CP, Ban K, Dujka ME, McConkey DJ, Munsell M, Palladino M, et al. NPI-0052, a novel proteasome inhibitor, induces caspase-8 and ROS-dependent apoptosis alone and in combination with HDAC inhibitors in leukemia cells. Blood. 2007;110:267–77.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Ling YH, Liebes L, Zou Y, Perez-Soler R. Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic response to Bortezomib, a novel proteasome inhibitor, in human H460 non-small cell lung cancer cells. J Biol Chem. 2003;278:33714–23.

    Article  PubMed  CAS  Google Scholar 

  37. Perez-Galan P, Roue G, Villamor N, Montserrat E, Campo E, Colomer D. The proteasome inhibitor bortezomib induces apoptosis in mantle-cell lymphoma through generation of ROS and Noxa activation independent of p53 status. Blood. 2006;107:257–64.

    Article  PubMed  CAS  Google Scholar 

  38. Weniger MA, Rizzatti EG, Perez-Galan P, Liu D, Wang Q, Munson PJ, et al. Treatment-induced oxidative stress and cellular antioxidant capacity determine response to bortezomib in mantle cell lymphoma. Clin Cancer Res. 2011;17:5101–12.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Wengrod J, Wang D, Weiss S, Zhong H, Osman I, Gardner LB. Phosphorylation of eIF2alpha triggered by mTORC1 inhibition and PP6C activation is required for autophagy and is aberrant in PP6C-mutated melanoma. Sci Signal. 2015;8:ra27.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. He Y, Correa AM, Raso MG, Hofstetter WL, Fang B, Behrens C, et al. The role of PKR/eIF2alpha signaling pathway in prognosis of non-small cell lung cancer. PLoS ONE. 2011;6:e24855.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Moreno JA, Radford H, Peretti D, Steinert JR, Verity N, Martin MG, et al. Sustained translational repression by eIF2alpha-P mediates prion neurodegeneration. Nature. 2012;485:507–11.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  42. Jousse C, Oyadomari S, Novoa I, Lu P, Zhang Y, Harding HP, et al. Inhibition of a constitutive translation initiation factor 2alpha phosphatase, CReP, promotes survival of stressed cells. J Cell Biol. 2003;163:767–75.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Kawazoe Y, Nakai A, Tanabe M, Nagata K. Proteasome inhibition leads to the activation of all members of the heat-shock-factor family. Eur J Biochem. 1998;255:356–62.

    Article  PubMed  CAS  Google Scholar 

  44. Bush KT, Goldberg AL, Nigam SK. Proteasome inhibition leads to a heat-shock response, induction of endoplasmic reticulum chaperones, and thermotolerance. J Biol Chem. 1997;272:9086–92.

    Article  PubMed  CAS  Google Scholar 

  45. Fribley A, Zeng Q, Wang CY. Proteasome inhibitor PS-341 induces apoptosis through induction of endoplasmic reticulum stress-reactive oxygen species in head and neck squamous cell carcinoma cells. Mol Cell Biol. 2004;24:9695–704.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Nawrocki ST, Carew JS, Dunner K Jr., Boise LH, Chiao PJ, Huang P, et al. Bortezomib inhibits PKR-like endoplasmic reticulum (ER) kinase and induces apoptosis via ER stress in human pancreatic cancer cells. Cancer Res. 2005;65:11510–9.

    Article  PubMed  CAS  Google Scholar 

  47. Nawrocki ST, Carew JS, Pino MS, Highshaw RA, Dunner K Jr, Huang P, et al. Bortezomib sensitizes pancreatic cancer cells to endoplasmic reticulum stress-mediated apoptosis. Cancer Res. 2005;65:11658–66.

    Article  PubMed  CAS  Google Scholar 

  48. Lu L, Han AP, Chen JJ. Translation initiation control by heme-regulated eukaryotic initiation factor 2alpha kinase in erythroid cells under cytoplasmic stresses. Mol Cell Biol. 2001;21:7971–80.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. Mittal SP, Kulkarni AP, Mathai J, Chattopadhyay S, Pal JK. Dose-dependent differential response of mammalian cells to cytoplasmic stress is mediated through the heme-regulated eIF2alpha kinase. Int J Biochem & Cell Biol. 2014;54:186–97.

    Article  CAS  Google Scholar 

  50. Rafie-Kolpin M, Han AP, Chen JJ. Autophosphorylation of threonine 485 in the activation loop is essential for attaining eIF2alpha kinase activity of HRI. Biochemistry. 2003;42:6536–44.

    Article  PubMed  CAS  Google Scholar 

  51. Matts RL, Xu Z, Pal JK, Chen JJ. Interactions of the heme-regulated eIF-2 alpha kinase with heat shock proteins in rabbit reticulocyte lysates. J Biol Chem. 1992;267:18160–7.

    PubMed  CAS  Google Scholar 

  52. Matts RL, Hurst R, Xu Z. Denatured proteins inhibit translation in hemin-supplemented rabbit reticulocyte lysate by inducing the activation of the heme-regulated eIF-2 alpha kinase. Biochemistry. 1993;32:7323–8.

    Article  PubMed  CAS  Google Scholar 

  53. Uma S, Thulasiraman V, Matts RL. Dual role for Hsc70 in the biogenesis and regulation of the heme-regulated kinase of the alpha subunit of eukaryotic translation initiation factor 2. Mol Cell Biol. 1999;19:5861–71.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  54. Suraweera A, Munch C, Hanssum A, Bertolotti A. Failure of amino acid homeostasis causes cell death following proteasome inhibition. Mol Cell. 2012;48:242–53.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  55. Fournier MJ, Gareau C, Mazroui R. The chemotherapeutic agent bortezomib induces the formation of stress granules. Cancer Cell Int. 2010;10:12.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  56. Burwick N, Zhang MY, de la Puente P, Azab AK, Hyun TS, Ruiz-Gutierrez M, et al. The eIF2-alpha kinase HRI is a novel therapeutic target in multiple myeloma. Leuk Res. 2017;55:23–32.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  57. Yefidoff-Freedman R, Fan J, Yan L, Zhang Q, Dos Santos GRR, Rana S, et al. Development of 1-((1,4-trans)-4-Aryloxycyclohexyl)-3-arylurea activators of heme-regulated inhibitor as selective activators of the eukaryotic initiation factor 2 alpha (eIF2alpha) phosphorylation arm of the integrated endoplasmic reticulum stress response. J Med Chem. 2017;60:5392–406.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from NIH/NCI (R01 CA127494), and the M.D. Anderson Cancer Center Support Grant P30 CA016672-38 (D.J.M.). M.C.W. would like to acknowledge awards from The Albert Schweitzer Fellowship and the Tzu-Chi Foundation

Author information

Author notes

  1. David J. McConkey

    Present address: Johns Hopkins Greenberg Bladder Cancer Institute, Baltimore, MD, 21287, USA

Authors and Affiliations

  1. Departments of Urology, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA

    Matthew C. White, Keyi Zhu, Katherine Xiong & David J. McConkey

  2. Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA

    Matthew C. White, Keyi Zhu, Katherine Xiong & David J. McConkey

  3. The Program in Experimental Therapeutics, The University of Texas Graduate School of Biomedical Sciences, Houston, TX, 77030, USA

    Matthew C. White, Rebecca D. Schroeder & David J. McConkey

Authors
  1. Matthew C. White
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rebecca D. Schroeder
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Keyi Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Katherine Xiong
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. David J. McConkey
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to David J. McConkey.

Ethics declarations

Conflict of interest

David McConkey holds stock options with ApoCell, Inc.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

White, M.C., Schroeder, R.D., Zhu, K. et al. HRI-mediated translational repression reduces proteotoxicity and sensitivity to bortezomib in human pancreatic cancer cells. Oncogene 37, 4413–4427 (2018). https://doi.org/10.1038/s41388-018-0227-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41388-018-0227-y

This article is cited by

Search

Advanced search

Quick links