Translational Therapeutics

Regulation of hypoxia-induced autophagy in glioblastoma involves ATG9A

  • British Journal of Cancer (2017) 117, 813825 (05 September 2017)
  • doi:10.1038/bjc.2017.263
  • Download Citation
Revised:
Accepted:
Published online:

Abstract

Background:

Hypoxia is negatively associated with glioblastoma (GBM) patient survival and contributes to tumour resistance. Anti-angiogenic therapy in GBM further increases hypoxia and activates survival pathways. The aim of this study was to determine the role of hypoxia-induced autophagy in GBM.

Methods:

Pharmacological inhibition of autophagy was applied in combination with bevacizumab in GBM patient-derived xenografts (PDXs). Sensitivity towards inhibitors was further tested in vitro under normoxia and hypoxia, followed by transcriptomic analysis. Genetic interference was done using ATG9A-depleted cells.

Results:

We find that GBM cells activate autophagy as a survival mechanism to hypoxia, although basic autophagy appears active under normoxic conditions. Although single agent chloroquine treatment in vivo significantly increased survival of PDXs, the combination with bevacizumab resulted in a synergistic effect at low non-effective chloroquine dose. ATG9A was consistently induced by hypoxia, and silencing of ATG9A led to decreased proliferation in vitro and delayed tumour growth in vivo. Hypoxia-induced activation of autophagy was compromised upon ATG9A depletion.

Conclusions:

This work shows that inhibition of autophagy is a promising strategy against GBM and identifies ATG9 as a novel target in hypoxia-induced autophagy. Combination with hypoxia-inducing agents may provide benefit by allowing to decrease the effective dose of autophagy inhibitors.

  • Subscribe to British Journal of Cancer for full access:

    $659

    Subscribe

Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

Change history

  • Corrected online 05 September 2017

References

  1. , , , , , , , , , , , , , (2013) Granzyme B degradation by autophagy decreases tumor cell susceptibility to natural killer-mediated lysis under hypoxia. Proc Natl Acad Sci USA 110: 17450–17455.

  2. , , , , , , (2009) Hypoxia-induced autophagy is mediated through hypoxia-inducible factor induction of BNIP3 and BNIP3L via their BH3 domains. Mol Cell Biol 29: 2570–2581.

  3. , , (2008) The impact of O2 availability on human cancer. Nat Rev Cancer 8: 967–975.

  4. , , , , , , , , , , , , , , , , , , (2016) Molecular crosstalk between tumour and brain parenchyma instructs histopathological features in glioblastoma. Oncotarget.

  5. , , , , , , (2016) Tyrosine kinase receptor EGFR regulates the switch in cancer cells between cell survival and cell death induced by autophagy in hypoxia. Autophagy 12: 1029–1046.

  6. , , , , (2008) Oxidative stress induces autophagic cell death independent of apoptosis in transformed and cancer cells. Cell Death Differ 15: 171–182.

  7. , , , (2013) Endoplasmic reticulum stress triggers autophagy in malignant glioma cells undergoing cyclosporine a-induced cell death. Oncogene 32: 1518–1529.

  8. , , , , , , , , , , , , , , , (2016) Excess sphingomyelin disturbs ATG9A trafficking and autophagosome closure. Autophagy 12: 833–849.

  9. , , , , , , , (2008) The genomic profile of human malignant glioma is altered early in primary cell culture and preserved in spheroids. Oncogene 27: 2091–2096.

  10. , , , , , , , , , (2015) Targeted proteomics to assess the response to anti-angiogenic treatment in human glioblastoma. Mol Cell Proteomics 15: 481–492.

  11. , , , , , , , , , , , , , (2004) Comparative measurements of hypoxia in human brain tumors using needle electrodes and EF5 binding. Cancer Res 64: 1886–1892.

  12. , , , , , , , , , , , , , , , , , , (2015) Bevacizumab treatment induces metabolic adaptation toward anaerobic metabolism in glioblastomas. Acta Neuropathol 129: 115–131.

  13. , , , , (2012) Chloroquine or chloroquine-PI3K/Akt pathway inhibitor combinations strongly promote gamma-irradiation-induced cell death in primary stem-like glioma cells. PloS ONE 7: e47357.

  14. , , , , , , , (2000) Genomic expression programs in the response of yeast cells to environmental changes. Mol Biol Cell 11: 4241–4257.

  15. (2016) Antiangiogenic therapy for glioblastoma: complex biology and complicated results. J Clin Oncol 34: 1567–1569.

  16. , , (2010) Autophagy: cellular and molecular mechanisms. J Pathol 221: 3–12.

  17. , , , , , , , , , (2013) Side population in human glioblastoma is non-tumorigenic and characterizes brain endothelial cells. Brain 136: 1462–1475.

  18. , , , , , (2010) Multicellular tumor spheroids: an underestimated tool is catching up again. J Biotechnol 148: 3–15.

  19. , , , , , , (2012) Hypoxia-induced autophagy promotes tumor cell survival and adaptation to antiangiogenic treatment in glioblastoma. Cancer Res 72: 1773–1783.

  20. , (2014) Transcriptional regulation of ATG9 by the Pho23-Rpd3 complex modulates the frequency of autophagosome formation. Autophagy 10: 1681–1682.

  21. , , , , , (2004) Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells. Cell Death Differ 11: 448–457.

  22. , , , , , , , , , , , , , , , , , (2011) Anti-VEGF treatment reduces blood supply and increases tumor cell invasion in glioblastoma. Proc Natl Acad Sci USA 108: 3749–3754.

  23. , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , (2016) Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12: 1–222.

  24. , , , , , , , (2003) BNIP3 plays a role in hypoxic cell death in human epithelial cells that is inhibited by growth factors EGF and IGF. Oncogene 22: 4734–4744.

  25. , , , , , , (2016) TBC1D14 regulates autophagy via the TRAPP complex and ATG9 traffic. EMBO J 35: 281–301.

  26. , , , , , , , , , , , , , , , , , , , , , , (2014) Tumor vessel normalization by chloroquine independent of autophagy. Cancer Cell 26: 190–206.

  27. , , , , , , , , , , , , , , , (2006) TRANSFAC and its module TRANSCompel: transcriptional gene regulation in eukaryotes. Nucleic Acids Res 34: D108–D110.

  28. , , , , , (2012) Chloroquine sensitizes breast cancer cells to chemotherapy independent of autophagy. Autophagy 8: 200–212.

  29. , (2009) Atypical BH3-domains of BNIP3 and BNIP3L lead to autophagy in hypoxia. Autophagy 5: 868–869.

  30. , (2010) Hypoxia-induced autophagy: cell death or cell survival? Curr Opin Cell Biol 22: 177–180.

  31. , , , , , , (2009) Genome-wide association of hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha DNA binding with expression profiling of hypoxia-inducible transcripts. J Biol Chem 284: 16767–16775.

  32. , , , , , , , , , , (2011) The acquisition of resistance to TNFalpha in breast cancer cells is associated with constitutive activation of autophagy as revealed by a transcriptome analysis using a custom microarray. Autophagy 7: 760–770.

  33. , (2015) MTOR, PIK3C3, and autophagy: signaling the beginning from the end. Autophagy 11: 2375–2376.

  34. , , , , , , , , , , (2008) A novel eGFP-expressing immunodeficient mouse model to study tumor-host interactions. FASEB J 22: 3120–3128.

  35. , , , , (2012) Hypoxia-induced autophagy: a new player in cancer immunotherapy? Autophagy 8: 704–706.

  36. , , , , , , , , , , (2011) Blocking hypoxia-induced autophagy in tumors restores cytotoxic T-cell activity and promotes regression. Cancer Res 71: 5976–5986.

  37. , , , , , , (2012) Dynamic and transient interactions of Atg9 with autophagosomes, but not membrane integration, are required for autophagy. Mol Biol Cell 23: 1860–1873.

  38. , , (2006) Hypoxia signalling in cancer and approaches to enforce tumour regression. Nature 441: 437–443.

  39. , (2010) Autophagy and metabolism. Science 330: 1344–1348.

  40. , (2012) Autophagy regulation through Atg9 traffic. J Cell Biol 198: 151–153.

  41. , , , , , , , , , , , , , , , , , , , (2014) A phase I/II trial of hydroxychloroquine in conjunction with radiation therapy and concurrent and adjuvant temozolomide in patients with newly diagnosed glioblastoma multiforme. Autophagy 10: 1359–1368.

  42. , , , , , , , , , , , , , (2010) The unfolded protein response protects human tumor cells during hypoxia through regulation of the autophagy genes MAP1LC3B and ATG5. J Clin Invest 120: 127–141.

  43. , , , , , , , , , (2015) Comprehensive analysis of glycolytic enzymes as therapeutic targets in the treatment of glioblastoma. PLoS One 10: e0123544.

  44. , , , , , , , , (2009) Inhibition of autophagy at a late stage enhances imatinib-induced cytotoxicity in human malignant glioma cells. Int J Cancer 124: 1060–1071.

  45. , (2004) Autophagy in health and disease: a double-edged sword. Science 306: 990–995.

  46. , (2016) Transcription factor HIF1A: downstream targets, associated pathways, polymorphic hypoxia response element (HRE) sites, and initiative for standardization of reporting in scientific literature. Tumour Biol 37: 14851–14861.

  47. , , (2006) Adding chloroquine to conventional treatment for glioblastoma multiforme: a randomized, double-blind, placebo-controlled trial. Ann Intern Med 144: 337–343.

  48. , (2007) Hypoxia in cancer: significance and impact on clinical outcome. Cancer Metastasis Rev 26: 225–239.

  49. , , , , , , (2014) Autophagic degradation of GZMB/granzyme B: a new mechanism of hypoxic tumor cell escape from natural killer cell-mediated lysis. Autophagy 10: 173–175.

  50. , , , , , , , , , (2014) Metabolic-stress-induced rearrangement of the 14-3-3zeta interactome promotes autophagy via a ULK1- and AMPK-regulated 14-3-3zeta interaction with phosphorylated Atg9. Mol Cell Biol 34: 4379–4388.

  51. , , , , , , , , (2012) Atg9 vesicles are an important membrane source during early steps of autophagosome formation. J Cell Biol 198: 219–233.

  52. , , , , (2012) Hypoxia and hypoxia-inducible factors in glioblastoma multiforme progression and therapeutic implications. Exp Cell Res 318: 2417–2426.

  53. , , , (2011) The role of autophagy in cancer: therapeutic implications. Mol Cancer Ther 10: 1533–1541.

  54. , , , , , , , (2008) Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia. J Biol Chem 283: 10892–10903.

Download references

Acknowledgements

We thank Morgane Sanzey, Virginie Baus, and Amandine Bernard for technical assistance. We thank Dr Christel Herold-Mende (Department of Neurosurgery, University of Heidelberg, Germany) and Dr Uros Rajcevic (National Institute of Biology, Ljubljana, Slovenia) for providing cell lines. This work was supported by the Luxembourg Institute of Health (LIH), the Fonds National de la Recherche (FNR) of Luxembourg (ESCAPE 784322 BM, AFR grand to SAAR and AD) and the Fondation Cancer Luxembourg (INVGBM).

Author information

Affiliations

  1. NorLux Neuro-Oncology Laboratory, Department of Oncology, Luxembourg Institute of Health, L-1526 Luxembourg City, Luxembourg

    • Siti Aminah Abdul Rahim
    • , Anne Dirkse
    • , Anais Oudin
    • , Anne Schuster
    • , Jill Bohler
    • , Vanessa Barthelemy
    • , Anna Golebiewska
    •  & Simone P Niclou
  2. Faculty of Science, Technology and Communication, University of Luxembourg, Esch-sur-Alzette L-4365, Luxembourg

    • Anne Dirkse
  3. Proteome and Genome Research Unit, Department of Oncology, Luxembourg Institute of Health, L-1526 Luxembourg City, Luxembourg

    • Arnaud Muller
    •  & Laurent Vallar
  4. Laboratory of Experimental Cancer Research, Department of Oncology, Luxembourg Institute of Health, L-1526 Luxembourg City, Luxembourg

    • Bassam Janji
  5. KG Jebsen Brain Tumour Research Center, Department of Biomedicine, University of Bergen, N-5019 Bergen, Norway

    • Simone P Niclou

Authors

  1. Search for Siti Aminah Abdul Rahim in:

  2. Search for Anne Dirkse in:

  3. Search for Anais Oudin in:

  4. Search for Anne Schuster in:

  5. Search for Jill Bohler in:

  6. Search for Vanessa Barthelemy in:

  7. Search for Arnaud Muller in:

  8. Search for Laurent Vallar in:

  9. Search for Bassam Janji in:

  10. Search for Anna Golebiewska in:

  11. Search for Simone P Niclou in:

Competing interests

The authors declare no conflict of interest.

Corresponding author

Correspondence to Simone P Niclou.

Supplementary information

This work is published under the standard license to publish agreement. After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution-NonCommercial-Share Alike 4.0 Unported License.

Supplementary Information accompanies this paper on British Journal of Cancer website (http://www.nature.com/bjc)

Creative Commons BY-NC-SAFrom twelve months after its original publication, this work is licensed under the Creative Commons Attribution-NonCommercial-Share Alike 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/4.0/