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ATF4 orchestrates a program of BH3-only protein expression in severe hypoxia

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Abstract

Intratumoral hypoxia is associated with poor prognosis, regardless of the mode of therapy. Cancer cells survive this condition through activating several adaptive signaling pathways, including the integrated stress response (ISR) and autophagy. Activating transcription factor 4 (ATF4) is the major transcriptional mediator of the ISR, which we have shown to be involved in autophagy regulation to protect cells from severe hypoxia. Here we demonstrate that ATF4 orchestrates a program of BH3-only protein expression in severe hypoxia. We find that the BH3-only proteins HRK, PUMA, and NOXA are transcriptionally induced in severe hypoxia and that their expression is abrogated by RNA interference against ATF4. In particular, we show that the BH3-only protein harakiri (HRK) is transactivated by ATF4 in severe hypoxia through direct binding of ATF4 to the promoter region. Furthermore, we demonstrate through siRNA knockdown that HRK induces autophagy and promotes cancer cell survival in severe hypoxia.

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References

  1. Thomlinson RH, Gray LH (1955) The histological structure of some human lung cancers and the possible implications for radiotherapy. Br J Cancer 9:539–549

    Article  PubMed  CAS  Google Scholar 

  2. Bertout JA, Patel SA, Simon MC (2008) The impact of O2 availability on human cancer. Nat Rev Cancer 8:967–975

    Article  PubMed  CAS  Google Scholar 

  3. Wouters BG, Koritzinsky M (2008) Hypoxia signalling through mTOR and the unfolded protein response in cancer. Nat Rev Cancer 8:851–864

    Article  PubMed  CAS  Google Scholar 

  4. Hockel M, Vorndran B, Schlenger K, Baussmann E, Knapstein PG (1993) Tumor oxygenation: a new predictive parameter in locally advanced cancer of the uterine cervix. Gynecol Oncol 51:141–149

    Article  PubMed  CAS  Google Scholar 

  5. Graeber TG, Osmanian C, Jacks T, Housman DE, Koch CJ et al (1996) Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours. Nature 379:88–91

    Article  PubMed  CAS  Google Scholar 

  6. Harris AL (2002) Hypoxia—a key regulatory factor in tumour growth. Nat Rev Cancer 2:38–47

    Article  PubMed  CAS  Google Scholar 

  7. Tu BP, Weissman JS (2002) The FAD- and O(2)-dependent reaction cycle of Ero1-mediated oxidative protein folding in the endoplasmic reticulum. Mol Cell 10:983–994

    Article  PubMed  CAS  Google Scholar 

  8. Harding HP, Zhang Y, Zeng H, Novoa I, Lu PD et al (2003) An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Mol Cell 11:619–633

    Article  PubMed  CAS  Google Scholar 

  9. Koritzinsky M, Magagnin MG, van den Beucken T, Seigneuric R, Savelkouls K et al (2006) Gene expression during acute and prolonged hypoxia is regulated by distinct mechanisms of translational control. EMBO J 25:1114–1125

    Article  PubMed  CAS  Google Scholar 

  10. Smirnova JB, Selley JN, Sanchez-Cabo F, Carroll K, Eddy AA et al (2005) Global gene expression profiling reveals widespread yet distinctive translational responses to different eukaryotic translation initiation factor 2B-targeting stress pathways. Mol Cell Biol 25:9340–9349

    Article  PubMed  CAS  Google Scholar 

  11. Blais JD, Filipenko V, Bi M, Harding HP, Ron D et al (2004) Activating transcription factor 4 is translationally regulated by hypoxic stress. Mol Cell Biol 24:7469–7482

    Article  PubMed  CAS  Google Scholar 

  12. Ameri K, Harris AL (2008) Activating transcription factor 4. Int J Biochem Cell Biol 40:14–21

    Article  PubMed  CAS  Google Scholar 

  13. Fels DR, Koumenis C (2006) The PERK/eIF2alpha/ATF4 module of the UPR in hypoxia resistance and tumor growth. Cancer Biol Ther 5:723–728

    PubMed  CAS  Google Scholar 

  14. Ma Y, Hendershot LM (2004) The role of the unfolded protein response in tumour development: friend or foe? Nat Rev Cancer 4:966–977

    Article  PubMed  CAS  Google Scholar 

  15. Heath-Engel HM, Chang NC, Shore GC (2008) The endoplasmic reticulum in apoptosis and autophagy: role of the BCL-2 protein family. Oncogene 27:6419–6433

    Article  PubMed  CAS  Google Scholar 

  16. Mizushima N (2010) The role of the Atg1/ULK1 complex in autophagy regulation. Curr Opin Cell Biol 22:132–139

    Article  PubMed  CAS  Google Scholar 

  17. Sowter HM, Ratcliffe PJ, Watson P, Greenberg AH, Harris AL (2001) HIF-1-dependent regulation of hypoxic induction of the cell death factors BNIP3 and NIX in human tumors. Cancer Res 61:6669–6673

    PubMed  CAS  Google Scholar 

  18. Tracy K, Dibling BC, Spike BT, Knabb JR, Schumacker P et al (2007) BNIP3 is an RB/E2F target gene required for hypoxia-induced autophagy. Mol Cell Biol 27:6229–6242

    Article  PubMed  CAS  Google Scholar 

  19. Rouschop KM, van den Beucken T, Dubois L, Niessen H, Bussink J et al (2009) 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

    Article  PubMed  Google Scholar 

  20. Rzymski T, Milani M, Pike L, Buffa F, Mellor HR et al (2010) Regulation of autophagy by ATF4 in response to severe hypoxia. Oncogene 29:4424–4435

    Article  PubMed  CAS  Google Scholar 

  21. Milani M, Rzymski T, Mellor HR, Pike L, Bottini A et al (2009) The role of ATF4 stabilization and autophagy in resistance of breast cancer cells treated with Bortezomib. Cancer Res 69:4415–4423

    Article  PubMed  CAS  Google Scholar 

  22. Liang XH, Jackson S, Seaman M, Brown K, Kempkes B et al (1999) Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature 402:672–676

    Article  PubMed  CAS  Google Scholar 

  23. Elgendy M, Sheridan C, Brumatti G, Martin SJ (2011) Oncogenic ras-induced expression of noxa and Beclin-1 promotes autophagic cell death and limits clonogenic survival. Mol Cell 42(1):23–35

    Google Scholar 

  24. Maiuri MC, Le Toumelin G, Criollo A, Rain JC, Gautier F et al (2007) Functional and physical interaction between Bcl-X(L) and a BH3-like domain in Beclin-1. EMBO J 26:2527–2539

    Article  PubMed  CAS  Google Scholar 

  25. Pattingre S, Tassa A, Qu X, Garuti R, Liang XH et al (2005) Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell 122:927–939

    Article  PubMed  CAS  Google Scholar 

  26. Yee KS, Wilkinson S, James J, Ryan KM, Vousden KH (2009) PUMA- and Bax-induced autophagy contributes to apoptosis. Cell Death Differ 16:1135–1145

    Article  PubMed  CAS  Google Scholar 

  27. Coultas L, Terzano S, Thomas T, Voss A, Reid K et al (2007) Hrk/DP5 contributes to the apoptosis of select neuronal populations but is dispensable for haematopoietic cell apoptosis. J Cell Sci 120:2044–2052

    Article  PubMed  CAS  Google Scholar 

  28. Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, Aliev G et al (2008) Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4:151–175

    PubMed  CAS  Google Scholar 

  29. Rzymski T, Harris AL (2007) The unfolded protein response and integrated stress response to anoxia. Clin Cancer Res 13:2537–2540

    Article  PubMed  CAS  Google Scholar 

  30. Mujcic H, Rzymski T, Rouschop KM, Koritzinsky M, Milani M et al (2009) Hypoxic activation of the unfolded protein response (UPR) induces expression of the metastasis-associated gene LAMP3. Radiother Oncol 92:450–459

    Article  PubMed  CAS  Google Scholar 

  31. Nelson DA, Tan TT, Rabson AB, Anderson D, Degenhardt K et al (2004) Hypoxia and defective apoptosis drive genomic instability and tumorigenesis. Genes Dev 18:2095–2107

    Article  PubMed  CAS  Google Scholar 

  32. Kim JY, Ahn HJ, Ryu JH, Suk K, Park JH (2004) BH3-only protein Noxa is a mediator of hypoxic cell death induced by hypoxia-inducible factor 1alpha. J Exp Med 199:113–124

    Article  PubMed  CAS  Google Scholar 

  33. Ishihara T, Hoshino T, Namba T, Tanaka K, Mizushima T (2007) Involvement of up-regulation of PUMA in non-steroidal anti-inflammatory drug-induced apoptosis. Biochem Biophys Res Commun 356:711–717

    Article  PubMed  CAS  Google Scholar 

  34. Galehdar Z, Swan P, Fuerth B, Callaghan SM, Park DS et al (2010) Neuronal apoptosis induced by endoplasmic reticulum stress is regulated by ATF4-CHOP-mediated induction of the Bcl-2 homology 3-only member PUMA. J Neurosci 30:16938–16948

    Article  PubMed  CAS  Google Scholar 

  35. Wang Q, Mora-Jensen H, Weniger MA, Perez-Galan P, Wolford C et al (2009) ERAD inhibitors integrate ER stress with an epigenetic mechanism to activate BH3-only protein NOXA in cancer cells. Proc Natl Acad Sci USA 106:2200–2205

    Article  PubMed  CAS  Google Scholar 

  36. Rzymski T, Milani M, Singleton DC, Harris AL (2009) Role of ATF4 in regulation of autophagy and resistance to drugs and hypoxia. Cell Cycle 8:3838–3847

    Article  PubMed  CAS  Google Scholar 

  37. Inohara N, Ding L, Chen S, Nunez G (1997) harakiri, a novel regulator of cell death, encodes a protein that activates apoptosis and interacts selectively with survival-promoting proteins Bcl-2 and Bcl-X(L). EMBO J 16:1686–1694

    Article  PubMed  CAS  Google Scholar 

  38. Wakabayashi T, Kosaka J, Hommura S (2002) Up-regulation of Hrk, a regulator of cell death, in retinal ganglion cells of axotomized rat retina. Neurosci Lett 318:77–80

    Article  PubMed  CAS  Google Scholar 

  39. Gurzov EN, Ortis F, Cunha DA, Gosset G, Li M et al (2009) Signaling by IL-1beta + IFN-gamma and ER stress converge on DP5/Hrk activation: a novel mechanism for pancreatic beta-cell apoptosis. Cell Death Differ 16:1539–1550

    Article  PubMed  CAS  Google Scholar 

  40. Young JE, Garden GA, Martinez RA, Tanaka F, Sandoval CM et al (2009) Polyglutamine-expanded androgen receptor truncation fragments activate a Bax-dependent apoptotic cascade mediated by DP5/Hrk. J Neurosci 29:1987–1997

    Article  PubMed  CAS  Google Scholar 

  41. Imaizumi K, Morihara T, Mori Y, Katayama T, Tsuda M et al (1999) The cell death-promoting gene DP5, which interacts with the BCL2 family, is induced during neuronal apoptosis following exposure to amyloid beta protein. J Biol Chem 274:7975–7981

    Article  PubMed  CAS  Google Scholar 

  42. Aoki S, Su Q, Li H, Nishikawa K, Ayukawa K et al (2002) Identification of an axotomy-induced glycosylated protein, AIGP1, possibly involved in cell death triggered by endoplasmic reticulum-Golgi stress. J Neurosci 22:10751–10760

    PubMed  CAS  Google Scholar 

  43. Duennwald ML, Lindquist S (2008) Impaired ERAD and ER stress are early and specific events in polyglutamine toxicity. Genes Dev 22:3308–3319

    Article  PubMed  CAS  Google Scholar 

  44. Hoozemans JJ, van Haastert ES, Nijholt DA, Rozemuller AJ, Eikelenboom P et al (2009) The unfolded protein response is activated in pretangle neurons in Alzheimer’s disease hippocampus. Am J Pathol 174:1241–1251

    Article  PubMed  CAS  Google Scholar 

  45. Lee do Y, Lee KS, Lee HJ, Kim do H, Noh YH et al (2010) Activation of PERK signaling attenuates Abeta-mediated ER stress. PLoS ONE 5:e10489

    Article  PubMed  Google Scholar 

  46. Shimoke K, Sasaya H, Ikeuchi T (2011) Analysis of the role of nerve growth factor in promoting cell survival during endoplasmic reticulum stress in PC12 cells. Methods Enzymol 490:53–70

    Article  PubMed  CAS  Google Scholar 

  47. Oyadomari S, Mori M (2004) Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ 11:381–389

    Article  PubMed  CAS  Google Scholar 

  48. Towers E, Gilley J, Randall R, Hughes R, Kristiansen M et al (2009) The proapoptotic dp5 gene is a direct target of the MLK-JNK-c-Jun pathway in sympathetic neurons. Nucleic Acids Res 37:3044–3060

    Article  PubMed  CAS  Google Scholar 

  49. van den Beucken T, Koritzinsky M, Niessen H, Dubois L, Savelkouls K et al (2009) Hypoxia-induced expression of carbonic anhydrase 9 is dependent on the unfolded protein response. J Biol Chem 284:24204–24212

    Article  PubMed  Google Scholar 

  50. Roybal CN, Hunsaker LA, Barbash O, Vander Jagt DL, Abcouwer SF (2005) The oxidative stressor arsenite activates vascular endothelial growth factor mRNA transcription by an ATF4-dependent mechanism. J Biol Chem 280:20331–20339

    Article  PubMed  CAS  Google Scholar 

  51. Raval RR, Lau KW, Tran MG, Sowter HM, Mandriota SJ et al (2005) Contrasting properties of hypoxia-inducible factor 1 (HIF-1) and HIF-2 in von Hippel-Lindau-associated renal cell carcinoma. Mol Cell Biol 25:5675–5686

    Article  PubMed  CAS  Google Scholar 

  52. Zhang H, Bosch-Marce M, Shimoda LA, Tan YS, Baek JH et al (2008) Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia. J Biol Chem 283:10892–10903

    Article  PubMed  CAS  Google Scholar 

  53. Mortensen M, Watson AS, Simon AK (2011) Lack of autophagy in the hematopoietic system leads to loss of hematopoietic stem cell function and dysregulated myeloid proliferation. Autophagy 7:1069–1070

    Google Scholar 

  54. Mortensen M, Soilleux EJ, Djordjevic G, Tripp R, Lutteropp M et al (2011) The autophagy protein Atg7 is essential for hematopoietic stem cell maintenance. J Exp Med 208:455–467

    Google Scholar 

  55. Degenhardt K, Mathew R, Beaudoin B, Bray K, Anderson D et al (2006) Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell 10:51–64

    Article  PubMed  CAS  Google Scholar 

  56. Karantza-Wadsworth V, Patel S, Kravchuk O, Chen G, Mathew R et al (2007) Autophagy mitigates metabolic stress and genome damage in mammary tumorigenesis. Genes Dev 21:1621–1635

    Article  PubMed  CAS  Google Scholar 

  57. Mathew R, Kongara S, Beaudoin B, Karp CM, Bray K et al (2007) Autophagy suppresses tumor progression by limiting chromosomal instability. Genes Dev 21:1367–1381

    Article  PubMed  CAS  Google Scholar 

  58. Mathew R, Karp CM, Beaudoin B, Vuong N, Chen G et al (2009) Autophagy suppresses tumorigenesis through elimination of p62. Cell 137:1062–1075

    Article  PubMed  CAS  Google Scholar 

  59. Kroemer G, Levine B (2008) Autophagic cell death: the story of a misnomer. Nat Rev Mol Cell Biol 9:1004–1010

    Article  PubMed  CAS  Google Scholar 

  60. Lenardo MJ, McPhee CK, Yu L (2009) Autophagic cell death. Methods Enzymol 453:17–31

    Article  PubMed  CAS  Google Scholar 

  61. Kroemer G, Marino G, Levine B (2010) Autophagy and the integrated stress response. Mol Cell 40:280–293

    Article  PubMed  CAS  Google Scholar 

  62. Yang S, Wang X, Contino G, Liesa M, Sahin E et al (2011) Pancreatic cancers require autophagy for tumor growth. Genes Dev 25:717–729

    Article  PubMed  CAS  Google Scholar 

  63. Guo JY, Chen HY, Mathew R, Fan J, Strohecker AM et al (2011) Activated Ras requires autophagy to maintain oxidative metabolism and tumorigenesis. Genes Dev 25:460–470

    Article  PubMed  CAS  Google Scholar 

  64. Nakamura M, Ishida E, Shimada K, Nakase H, Sakaki T et al (2005) Frequent HRK inactivation associated with low apoptotic index in secondary glioblastomas. Acta Neuropathol 110:402–410

    Article  PubMed  CAS  Google Scholar 

  65. Nakamura M, Shimada K, Konishi N (2008) The role of HRK gene in human cancer. Oncogene 27(Suppl 1):S105–S113

    Article  PubMed  CAS  Google Scholar 

  66. Obata T, Toyota M, Satoh A, Sasaki Y, Ogi K et al (2003) Identification of HRK as a target of epigenetic inactivation in colorectal and gastric cancer. Clin Cancer Res 9:6410–6418

    PubMed  CAS  Google Scholar 

  67. Yu J, Zhang L, Hwang PM, Kinzler KW, Vogelstein B (2001) PUMA induces the rapid apoptosis of colorectal cancer cells. Mol Cell 7:673–682

    Article  PubMed  CAS  Google Scholar 

  68. Li J, Lee B, Lee AS (2006) Endoplasmic reticulum stress-induced apoptosis: multiple pathways and activation of p53-up-regulated modulator of apoptosis (PUMA) and NOXA by p53. J Biol Chem 281:7260–7270

    Article  PubMed  CAS  Google Scholar 

  69. Rzymski T, Paantjens A, Bod J, Harris AL (2008) Multiple pathways are involved in the anoxia response of SKIP3 including HuR-regulated RNA stability, NF-kappaB and ATF4. Oncogene 27:4532–4543

    Article  PubMed  CAS  Google Scholar 

  70. Otsuki Y, Li Z, Shibata MA (2003) Apoptotic detection methods–from morphology to gene. Prog Histochem Cytochem 38:275–339

    Article  PubMed  Google Scholar 

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Acknowledgments

Funding from Cancer Research UK, the Rhodes Trust, and the Natural Sciences and Engineering Research Council of Canada supported this work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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The authors declare no competing interests, financial or otherwise.

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Authors and Affiliations

  1. Growth Factor Group, Cancer Research UK, Molecular Oncology Laboratories, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Headington, Oxford, OX3 9DS, UK

    Luke R. G. Pike & Adrian L. Harris

  2. Nuffield Department of Medicine, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK

    Kanchan Phadwal & Anna Katharina Simon

  3. BRC Translational Immunology BRC lab, NIHR, Oxford, UK

    Kanchan Phadwal & Anna Katharina Simon

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  1. Luke R. G. Pike
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  2. Kanchan Phadwal
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  4. Adrian L. Harris
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Correspondence to Adrian L. Harris.

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Pike, L.R.G., Phadwal, K., Simon, A.K. et al. ATF4 orchestrates a program of BH3-only protein expression in severe hypoxia. Mol Biol Rep 39, 10811–10822 (2012). https://doi.org/10.1007/s11033-012-1975-3

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  • DOI: https://doi.org/10.1007/s11033-012-1975-3

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