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.

  • Original Article
  • Published:

Inhibition of the mitochondrial pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase by doxorubicin and brequinar sensitizes cancer cells to TRAIL-induced apoptosis

Oncogene volume 33, pages 3538–3549 (2014)Cite this article

Subjects

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent in selectively killing tumor cells. However, TRAIL monotherapy has not been successful as many cancer cells are resistant to TRAIL. Chemotherapeutic agents, such as doxorubicin have been shown to act synergistically with TRAIL, but the exact mechanisms of actions are poorly understood. In this study, we performed high-throughput small interfering RNA screening and genome-wide gene expression profiling on doxorubicin-treated U1690 cells to explore novel mechanisms underlying doxorubicin-TRAIL synergy. The screening and expression profiling results were integrated and dihydroorotate dehydrogenase (DHODH) was identified as a potential candidate. DHODH is the rate-limiting enzyme in the pyrimidine synthesis pathway, and its expression was downregulated by doxorubicin. We demonstrated that silencing of DHODH or inhibition of DHODH activity by brequinar dramatically increased the sensitivity of U1690 cells to TRAIL-induced apoptosis both in 2D and 3D cultures, and was accompanied by downregulation of c-FLIPL as well as by mitochondrial depolarization. In addition, uridine, an end product of the pyrimidine synthesis pathway was able to rescue the sensitization effects initiated by both brequinar and doxorubicin. Furthermore, several other cancer cell lines, LNCaP, MCF-7 and HT-29 were also shown to be sensitized to TRAIL by brequinar. Taken together, our findings have identified a novel protein target and its inhibitor, brequinar, as a potential agent in TRAIL-based combinatorial cancer therapy and highlighted for the first time the importance of mitochondrial DHODH enzyme and pyrimidine pathway in mediating TRAIL sensitization in cancer cells.

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

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8

Similar content being viewed by others

Accession codes

Accessions

Gene Expression Omnibus

References

  1. Johnstone RW, Frew AJ, Smyth MJ . The TRAIL apoptotic pathway in cancer onset, progression and therapy. Nat Rev Cancer 2008; 8: 782–798.

    Article  CAS  PubMed  Google Scholar 

  2. Ashkenazi A, Dixit VM . Apoptosis control by death and decoy receptors. Curr Opin Cell Biol 1999; 11: 255–260.

    Article  CAS  PubMed  Google Scholar 

  3. Falschlehner C, Emmerich CH, Gerlach B, Walczak H . TRAIL signalling: decisions between life and death. Int J Biochem Cell Biol 2007; 39: 1462–1475.

    Article  CAS  PubMed  Google Scholar 

  4. Pitti RM, Marsters SA, Ruppert S, Donahue CJ, Moore A, Ashkenazi A . Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family. J Biol Chem 1996; 271: 12687–12690.

    Article  CAS  PubMed  Google Scholar 

  5. Wiley SR, Schooley K, Smolak PJ, Din WS, Huang CP, Nicholl JK et al. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 1995; 3: 673–682.

    Article  CAS  PubMed  Google Scholar 

  6. Ashkenazi A, Pai RC, Fong S, Leung S, Lawrence DA, Marsters SA et al. Safety and antitumor activity of recombinant soluble Apo2 ligand. J Clin Invest 1999; 104: 155–162.

    CAS  PubMed  PubMed Central  Google Scholar 

  7. LeBlanc HN, Ashkenazi A . Apo2L/TRAIL and its death and decoy receptors. Cell Death Differ 2003; 10: 66–75.

    Article  CAS  PubMed  Google Scholar 

  8. Koschny R, Walczak H, Ganten TM . The promise of TRAIL--potential and risks of a novel anticancer therapy. J Mol Med 2007; 85: 923–935.

    Article  CAS  PubMed  Google Scholar 

  9. Vaculova A, Kaminskyy V, Jalalvand E, Surova O, Zhivotovsky B . Doxorubicin and etoposide sensitize small cell lung carcinoma cells expressing caspase-8 to TRAIL. Mol Cancer 2010; 9: 87.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Shivapurkar N, Reddy J, Matta H, Sathyanarayana UG, Huang CX, Toyooka S et al. Loss of expression of death-inducing signaling complex (DISC) components in lung cancer cell lines and the influence of MYC amplification. Oncogene 2002; 21: 8510–8514.

    Article  CAS  PubMed  Google Scholar 

  11. Hopkins-Donaldson S, Ziegler A, Kurtz S, Bigosch C, Kandioler D, Ludwig C et al. Silencing of death receptor and caspase-8 expression in small cell lung carcinoma cell lines and tumors by DNA methylation. Cell Death Differ 2003; 10: 356–364.

    Article  CAS  PubMed  Google Scholar 

  12. Kaminskyy VO, Surova OV, Piskunova T, Zborovskaya IB, Tchevkina EM, Andera L et al. Upregulation of c-FLIP-short in response to TRAIL promotes survival of NSCLC cells, which could be suppressed by inhibition of Ca2+/calmodulin signaling. Cell Death Dis 2013; 4: e522.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Mahmood Z, Shukla Y . Death receptors: targets for cancer therapy. Exp Cell Res 2010; 316: 887–899.

    Article  CAS  PubMed  Google Scholar 

  14. Papenfuss K, Cordier SM, Walczak H . Death receptors as targets for anti-cancer therapy. J Cell Mol Med 2008; 12: 2566–2585.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Mellier G, Huang S, Shenoy K, Pervaiz S . TRAILing death in cancer. Mol Aspects Med 2010; 31: 93–112.

    Article  CAS  PubMed  Google Scholar 

  16. Liu S, Neidhardt EA, Grossman TH, Ocain T, Clardy J . Structures of human dihydroorotate dehydrogenase in complex with antiproliferative agents. Structure 2000; 8: 25–33.

    Article  CAS  PubMed  Google Scholar 

  17. Loffler M, Fairbanks LD, Zameitat E, Marinaki AM, Simmonds HA . Pyrimidine pathways in health and disease. Trends Mol Med 2005; 11: 430–437.

    Article  PubMed  Google Scholar 

  18. Vyas VK, Ghate M . Recent developments in the medicinal chemistry and therapeutic potential of dihydroorotate dehydrogenase (DHODH) inhibitors. Mini Rev Med Chem 2011; 11: 1039–1055.

    Article  CAS  PubMed  Google Scholar 

  19. Cramer DV . Brequinar sodium. Pediatr Nephrol 1995; 9 (Suppl): S52–S55.

    Article  PubMed  Google Scholar 

  20. Peters GJ, Nadal JC, Laurensse EJ, de Kant E, Pinedo HM . Retention of in vivo antipyrimidine effects of Brequinar sodium (DUP-785; NSC 368390) in murine liver, bone marrow and colon cancer. Biochem Pharmacol 1990; 39: 135–144.

    Article  CAS  PubMed  Google Scholar 

  21. Kojima Y, Nakayama M, Nishina T, Nakano H, Koyanagi M, Takeda K et al. Importin beta1 protein-mediated nuclear localization of death receptor 5 (DR5) limits DR5/tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced cell death of human tumor cells. J Biol Chem 2011; 286: 43383–43393.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kawasumi M, Nghiem P . Chemical genetics: elucidating biological systems with small-molecule compounds. J Invest Dermatol 2007; 127: 1577–1584.

    Article  CAS  PubMed  Google Scholar 

  23. Coussens MJ, Corman C, Fischer AL, Sago J, Swarthout J . MISSION LentiPlex pooled shRNA library screening in mammalian cells. J Vis Exp 2011. 3305.

  24. Aza-Blanc P, Cooper CL, Wagner K, Batalov S, Deveraux QL, Cooke MP . Identification of modulators of TRAIL-induced apoptosis via RNAi-based phenotypic screening. Mol Cell 2003; 12: 627–637.

    Article  CAS  PubMed  Google Scholar 

  25. Ovcharenko D, Kelnar K, Johnson C, Leng N, Brown D . Genome-scale microRNA and small interfering RNA screens identify small RNA modulators of TRAIL-induced apoptosis pathway. Cancer Res 2007; 67: 10782–10788.

    Article  CAS  PubMed  Google Scholar 

  26. Booth NL, Sayers TJ, Brooks AD, Thomas CL, Jacobsen K, Goncharova EI et al. A cell-based high-throughput screen to identify synergistic TRAIL sensitizers. Cancer Immunol Immunother 2009; 58: 1229–1244.

    Article  PubMed  Google Scholar 

  27. Finlay D, Richardson RD, Landberg LK, Howes AL, Vuori K, Novel HTS . Strategy identifies TRAIL-sensitizing compounds acting specifically through the caspase-8 apoptotic axis. PLoS One 2010; 5: e13375.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Taylor DJ, Parsons CE, Han H, Jayaraman A, Rege K . Parallel screening of FDA-approved antineoplastic drugs for identifying sensitizers of TRAIL-induced apoptosis in cancer cells. BMC Cancer 2011; 11: 470.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Evans ME, Jones DP, Ziegler TR . Glutamine inhibits cytokine-induced apoptosis in human colonic epithelial cells via the pyrimidine pathway. Am J Physiol Gastrointest Liver Physiol 2005; 289: G388–G396.

    Article  CAS  PubMed  Google Scholar 

  30. Shawver LK, Schwartz DP, Mann E, Chen H, Tsai J, Chu L et al. Inhibition of platelet-derived growth factor-mediated signal transduction and tumor growth by N-[4-(trifluoromethyl)-phenyl]5-methylisoxazole-4-carboxamide. Clin Cancer Res 1997; 3: 1167–1177.

    CAS  PubMed  Google Scholar 

  31. Merrill JE, Hanak S, Pu SF, Liang J, Dang C, Iglesias-Bregna D et al. Teriflunomide reduces behavioral, electrophysiological, and histopathological deficits in the dark Agouti rat model of experimental autoimmune encephalomyelitis. J Neurol 2009; 256: 89–103.

    Article  CAS  PubMed  Google Scholar 

  32. Kaminskyy VO, Surova OV, Vaculova A, Zhivotovsky B . Combined inhibition of DNA methyltransferase and histone deacetylase restores caspase-8 expression and sensitizes SCLC cells to TRAIL. Carcinogenesis 2011; 32: 1450–1458.

    Article  CAS  PubMed  Google Scholar 

  33. White RM, Cech J, Ratanasirintrawoot S, Lin CY, Rahl PB, Burke CJ et al. DHODH modulates transcriptional elongation in the neural crest and melanoma. Nature 2011; 471: 518–522.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. O’Donnell EF, Saili KS, Koch DC, Kopparapu PR, Farrer D, Bisson WH et al. The anti-inflammatory drug leflunomide is an agonist of the aryl hydrocarbon receptor. PLoS One 2012; 5: e13128.

    Article  Google Scholar 

  35. Baumann P, Mandl-Weber S, Volkl A, Adam C, Bumeder I, Oduncu F et al. Dihydroorotate dehydrogenase inhibitor A771726 (leflunomide) induces apoptosis and diminishes proliferation of multiple myeloma cells. Mol Cancer Ther 2009; 8: 366–375.

    Article  CAS  PubMed  Google Scholar 

  36. Tang X, Yang J, Li J . Sensitization of human hepatic stellate cells to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by leflunomide. Biol Pharm Bull 2009; 32: 963–967.

    Article  CAS  PubMed  Google Scholar 

  37. Cleaveland ES, Zaharevitz DW, Kelley JA, Paull K, Cooney DA, Ford H Jr . Identification of a novel inhibitor (NSC 665564) of dihydroorotate dehydrogenase with a potency equivalent to brequinar. Biochem Biophys Res Commun 1996; 223: 654–659.

    Article  CAS  PubMed  Google Scholar 

  38. Maroun J, Ruckdeschel J, Natale R, Morgan R, Dallaire B, Sisk R et al. Multicenter phase II study of brequinar sodium in patients with advanced lung cancer. Cancer Chemother Pharmacol 1993; 32: 64–66.

    Article  CAS  PubMed  Google Scholar 

  39. c-FLIP Safa AR . A master anti-apoptotic regulator. Exp Oncol 2012; 34: 176–184.

    Google Scholar 

  40. Lee SC, Cheong HJ, Kim SJ, Yoon J, Kim HJ, Kim KH et al. Low-dose combinations of LBH589 and TRAIL can overcome TRAIL-resistance in colon cancer cell lines. Anticancer Res 2011; 31: 3385–3394.

    CAS  PubMed  Google Scholar 

  41. Zhu H, Zhao F, Yu S, He J, Deng L, Yi C et al. The synergistic effects of low-dose irinotecan and TRAIL on TRAIL-resistant HT-29 colon carcinoma in vitro and in vivo. Int J Mol Med 2012; 30: 1087–1094.

    Article  CAS  PubMed  Google Scholar 

  42. Kisim A, Atmaca H, Cakar B, Karabulut B, Sezgin C, Uzunoglu S et al. Pretreatment with AT-101 enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis of breast cancer cells by inducing death receptors 4 and 5 protein levels. J Cancer Res Clin Oncol 2012; 138: 1155–1163.

    Article  CAS  PubMed  Google Scholar 

  43. Yan S, Qu X, Xu C, Zhu Z, Zhang L, Xu L et al. Downregulation of Cbl-b by bufalin results in upregulation of DR4/DR5 and sensitization of TRAIL-induced apoptosis in breast cancer cells. J Cancer Res Clin Oncol 2012; 138: 1279–1289.

    Article  CAS  PubMed  Google Scholar 

  44. Szliszka E, Zydowicz G, Mizgala E, Krol W, Artepillin C . (3,5-Diprenyl-4-hydroxycinnamic acid) sensitizes LNCaP prostate cancer cells to TRAIL-induced apoptosis. Int J Oncol 2012; 41: 818–828.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Lee YJ, Seol JW, Jeong JK, Moon MH, Park SY . Inhibition of the ubiquitin-proteasome system sensitizes TRAIL-resistant prostate cancer cells by upregulation of death receptor 5. Mol Med Report 2011; 4: 1255–1259.

    CAS  Google Scholar 

  46. Harma V, Virtanen J, Makela R, Happonen A, Mpindi JP, Knuuttila M et al. A comprehensive panel of three-dimensional models for studies of prostate cancer growth, invasion and drug responses. PLoS One 2010; 5: e10431.

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We acknowledge Jouni Latoniitty and Rami Mäkelä for robotic technical assistance, Ville Härmä and Johannes Virtanen for advices and help in 3D experiments, Pirjo Käpylä for excellent technical support and Drs I Lavrik and P Krammer for antibodies. Finnish DNA Microarray Centre is acknowledged for performing TaqMan qRT–PCR and Illumina gene expression analysis. This work was supported by EU-APOSYS project (contract HEALTH-F4-2007-200767), EU (CHEMORES) project, the Swedish Research Foundation, the Swedish and Stockholm Cancer Foundations, and the Swedish Childhood Cancer Foundation.

Author information

Authors and Affiliations

  1. Medical Biotechnology, VTT Technical Research Centre of Finland, Turku, Finland

    T He, S Haapa-Paananen, P Kohonen, V Fey, O Kallioniemi & M Perälä

  2. Division of Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden

    V O Kaminskyy & B Zhivotovsky

  3. Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden

    P Kohonen

  4. FIMM-Institute for Molecular Medicine Finland, University of Helsinki, Helsinki, Finland

    O Kallioniemi

Authors
  1. T He
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S Haapa-Paananen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. V O Kaminskyy
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. P Kohonen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. V Fey
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. B Zhivotovsky
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. O Kallioniemi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. M Perälä
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to T He.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies this paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

He, T., Haapa-Paananen, S., Kaminskyy, V. et al. Inhibition of the mitochondrial pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase by doxorubicin and brequinar sensitizes cancer cells to TRAIL-induced apoptosis. Oncogene 33, 3538–3549 (2014). https://doi.org/10.1038/onc.2013.313

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2013.313

Keywords

This article is cited by

Search

Advanced search

Quick links