Tumor Biology

, Volume 33, Issue 1, pp 85–94 | Cite as

Cetuximab induces mitochondrial translocalization of EGFRvIII, but not EGFR: involvement of mitochondria in tumor drug resistance?

  • Agnieszka Dreier
  • Stefan Barth
  • Anand Goswami
  • Joachim Weis
Research Article


Dysregulation of growth factor receptors such as the epidermal growth factor receptor (EGFR) and of its truncated form EGFRvIII is frequently found in human tumors. EGFRvIII is a promising target for selective molecular tumor therapy because it is exclusively expressed by tumor cells. Cetuximab/Erbitux is a monoclonal antibody which targets EGFR and EGFRvIII. The effects of cetuximab on EGFRvIII but still the exact function and mechanism of cetuximab in relation to EGFR and EGFRvIII are incompletely understood. Therefore, we investigated the influence of cetuximab on EGFRvIII signaling and cellular survival. We found that cetuximab leads to increased internalization of EGFRvIII in NR6M cells but is unable to inhibit neither the activation of EGFRvIII nor its downstream signaling pathways. Incubation with cetuximab also did not alter the survival and proliferation of EGFRvIII-expressing cells. However, it caused increased mitochondrial activity and an increase in co-localization of EGFRvIII with mitochondria. These results demonstrate that interaction of EGFRvIII with mitochondria could play a role in survival of cetuximab-treated NR6M cells. Thus, a role of mitochondria in resistance to cetuximab has to be considered.


EGFR EGFRvIII Cetuximab Mitochondria Tumor drug resistance 



Anti-apoptotic kinase






Cytochrome C


Epidermal growth factor


Epidermal growth factor receptor (wild type receptor)


EGFR variant three (mutant receptor)


Extracellular signal-regulated kinase


Mitogen-activated protein kinase


Signal transducer and activator of transcription


Transforming growth factor alpha


Subunit of mitochondrial inner membrane translocase complex



We thank Dr. D. Bigner, Duke University, Durham, NC, USA, for supplying the NR6, NR6W, and NR6M cells; Dr. B. Lüscher, Institute of Biochemistry; and Dr. S. Arnold, Institute of Neuroanatomy, both RWTH Aachen University, for helpful discussions, and PD Dr. G. Brook, Institute of Neuropathology, RWTH Aachen University, for reading the manuscript.


This study was funded by the START program “Molecular tumor markers and their function,” Medical Faculty, RWTH Aachen University, Aachen, Germany.

Conflicts of interest


Supplementary material

13277_2011_248_MOESM1_ESM.pdf (5.2 mb)
ESM 1 (PDF 5.18 mb)


  1. 1.
    Wells A. EGF receptor. Int J Biochem Cell Biol. 1999;31(6):637–43.PubMedCrossRefGoogle Scholar
  2. 2.
    Harris RC, Chung E, Coffey RJ. EGF receptor ligands. Exp Cell Res. 2003;284(1):2–13.PubMedCrossRefGoogle Scholar
  3. 3.
    Singh AB, Harris RC. Autocrine, paracrine and juxtacrine signaling by EGFR ligands. Cell Signal. 2005;17(10):1183–93. doi: 10.1016/j.cellsig.2005.03.026.PubMedCrossRefGoogle Scholar
  4. 4.
    Prenzel N, Fischer OM, Streit S, Hart S, Ullrich A. The epidermal growth factor receptor family as a central element for cellular signal transduction and diversification. Endocr Relat Cancer. 2001;8(1):11–31.PubMedCrossRefGoogle Scholar
  5. 5.
    Brantley EC, Benveniste EN. Signal transducer and activator of transcription-3: a molecular hub for signaling pathways in gliomas. Mol Cancer Res. 2008;6(5):675–84. doi: 10.1158/1541-7786.MCR-07-2180.PubMedCrossRefGoogle Scholar
  6. 6.
    Pedersen MW, Meltorn M, Damstrup L, Poulsen HS. The type III epidermal growth factor receptor mutation. Biological significance and potential target for anti-cancer therapy. Ann Oncol. 2001;12(6):745–60.PubMedCrossRefGoogle Scholar
  7. 7.
    Humphrey PA, Wong AJ, Vogelstein B, Zalutsky MR, Fuller GN, Archer GE, et al. Anti-synthetic peptide antibody reacting at the fusion junction of deletion-mutant epidermal growth factor receptors in human glioblastoma. Proc Natl Acad Sci U S A. 1990;87(11):4207–11.PubMedCrossRefGoogle Scholar
  8. 8.
    Sugawa N, Ekstrand AJ, James CD, Collins VP. Identical splicing of aberrant epidermal growth factor receptor transcripts from amplified rearranged genes in human glioblastomas. Proc Natl Acad Sci U S A. 1990;87(21):8602–6.PubMedCrossRefGoogle Scholar
  9. 9.
    Bigner SH, Humphrey PA, Wong AJ, Vogelstein B, Mark J, Friedman HS, et al. Characterization of the epidermal growth factor receptor in human glioma cell lines and xenografts. Cancer Res. 1990;50(24):8017–22.PubMedGoogle Scholar
  10. 10.
    Yamazaki H, Ohba Y, Tamaoki N, Shibuya M. A deletion mutation within the ligand binding domain is responsible for activation of epidermal growth factor receptor gene in human brain tumors. Jpn J Cancer Res. 1990;81(8):773–9.PubMedCrossRefGoogle Scholar
  11. 11.
    Nagane M, Coufal F, Lin H, Bogler O, Cavenee WK, Huang HJ. A common mutant epidermal growth factor receptor confers enhanced tumorigenicity on human glioblastoma cells by increasing proliferation and reducing apoptosis. Cancer Res. 1996;56(21):5079–86.PubMedGoogle Scholar
  12. 12.
    Imai K, Takaoka A. Comparing antibody and small-molecule therapies for cancer. Nat Rev Cancer. 2006;6(9):714–27. doi: 10.1038/nrc1913.PubMedCrossRefGoogle Scholar
  13. 13.
    Veale D, Kerr N, Gibson GJ, Kelly PJ, Harris AL. The relationship of quantitative epidermal growth factor receptor expression in non-small cell lung cancer to long term survival. Br J Cancer. 1993;68(1):162–5.PubMedCrossRefGoogle Scholar
  14. 14.
    Libermann TA, Razon N, Bartal AD, Yarden Y, Schlessinger J, Soreq H. Expression of epidermal growth factor receptors in human brain tumors. Cancer Res. 1984;44(2):753–60.PubMedGoogle Scholar
  15. 15.
    Hendler FJ, Ozanne BW. Human squamous cell lung cancers express increased epidermal growth factor receptors. J Clin Invest. 1984;74(2):647–51. doi: 10.1172/JCI111463.PubMedCrossRefGoogle Scholar
  16. 16.
    Neal DE, Marsh C, Bennett MK, Abel PD, Hall RR, Sainsbury JR, et al. Epidermal-growth-factor receptors in human bladder cancer: comparison of invasive and superficial tumours. Lancet. 1985;1(8425):366–8.PubMedCrossRefGoogle Scholar
  17. 17.
    Moscatello DK, Holgado-Madruga M, Godwin AK, Ramirez G, Gunn G, Zoltick PW, et al. Frequent expression of a mutant epidermal growth factor receptor in multiple human tumors. Cancer Res. 1995;55(23):5536–9.PubMedGoogle Scholar
  18. 18.
    Voldborg BR, Damstrup L, Spang-Thomsen M, Poulsen HS. Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials. Ann Oncol. 1997;8(12):1197–206.PubMedCrossRefGoogle Scholar
  19. 19.
    Mendelsohn J, Baselga J. The EGF receptor family as targets for cancer therapy. Oncogene. 2000;19(56):6550–65. doi: 10.1038/sj.onc.1204082.PubMedCrossRefGoogle Scholar
  20. 20.
    Aerts HJ, Dubois L, Hackeng TM, Straathof R, Chiu RK, Lieuwes NG, et al. Development and evaluation of a cetuximab-based imaging probe to target EGFR and EGFRvIII. Radiother Oncol. 2007;83(3):326–32. doi: 10.1016/j.radonc.2007.04.030.PubMedCrossRefGoogle Scholar
  21. 21.
    Li S, Schmitz KR, Jeffrey PD, Wiltzius JJ, Kussie P, Ferguson KM. Structural basis for inhibition of the epidermal growth factor receptor by cetuximab. Cancer Cell. 2005;7(4):301–11. doi: 10.1016/j.ccr.2005.03.003.PubMedCrossRefGoogle Scholar
  22. 22.
    Harding J, Burtness B. Cetuximab: an epidermal growth factor receptor chemeric human-murine monoclonal antibody. Drugs Today (Barc). 2005;41(2):107–27. doi: 10.1358/dot.2005.41.2.882662.CrossRefGoogle Scholar
  23. 23.
    Jutten B, Dubois L, Li Y, Aerts H, Wouters BG, Lambin P, et al. Binding of cetuximab to the EGFRvIII deletion mutant and its biological consequences in malignant glioma cells. Radiother Oncol. 2009;92(3):393–8. doi: 10.1016/j.radonc.2009.06.021.PubMedCrossRefGoogle Scholar
  24. 24.
    Patel D, Lahiji A, Patel S, Franklin M, Jimenez X, Hicklin DJ, et al. Monoclonal antibody cetuximab binds to and down-regulates constitutively activated epidermal growth factor receptor vIII on the cell surface. Anticancer Res. 2007;27(5A):3355–66.PubMedGoogle Scholar
  25. 25.
    Fukai J, Nishio K, Itakura T, Koizumi F. Antitumor activity of cetuximab against malignant glioma cells overexpressing EGFR deletion mutant variant III. Cancer Sci. 2008;99(10):2062–9. doi: 10.1111/j.1349-7006.2008.00945.x.PubMedGoogle Scholar
  26. 26.
    Kampmeier F, Ribbert M, Nachreiner T, Dembski S, Beaufils F, Brecht A, et al. Site-specific, covalent labeling of recombinant antibody fragments via fusion to an engineered version of 6-O-alkylguanine DNA alkyltransferase. Bioconjug Chem. 2009;20(5):1010–5. doi: 10.1021/bc9000257.PubMedCrossRefGoogle Scholar
  27. 27.
    Batra SK, Castelino-Prabhu S, Wikstrand CJ, Zhu X, Humphrey PA, Friedman HS, et al. Epidermal growth factor ligand-independent, unregulated, cell-transforming potential of a naturally occurring human mutant EGFRvIII gene. Cell Growth Differ. 1995;6(10):1251–9.PubMedGoogle Scholar
  28. 28.
    Pruss RM, Herschman HR. Variants of 3 T3 cells lacking mitogenic response to epidermal growth factor. Proc Natl Acad Sci U S A. 1977;74(9):3918–21.PubMedCrossRefGoogle Scholar
  29. 29.
    Kampmeier F, Niesen J, Koers A, Ribbert M, Brecht A, Fischer R, et al. Rapid optical imaging of EGF receptor expression with a single-chain antibody SNAP-tag fusion protein. Eur J Nucl Med Mol Imaging. 2010;37(10):1926–34. doi: 10.1007/s00259-010-1482-5.PubMedCrossRefGoogle Scholar
  30. 30.
    O’Brien J, Wilson I, Orton T, Pognan F. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem. 2000;267(17):5421–6.PubMedCrossRefGoogle Scholar
  31. 31.
    Kawamoto T, Sato JD, Le A, Polikoff J, Sato GH, Mendelsohn J. Growth stimulation of A431 cells by epidermal growth factor: identification of high-affinity receptors for epidermal growth factor by an anti-receptor monoclonal antibody. Proc Natl Acad Sci U S A. 1983;80(5):1337–41.PubMedCrossRefGoogle Scholar
  32. 32.
    Nicholas MK, Lukas RV, Jafri NF, Faoro L, Salgia R. Epidermal growth factor receptor - mediated signal transduction in the development and therapy of gliomas. Clin Cancer Res. 2006;12(24):7261–70. doi: 10.1158/1078-0432.CCR-06-0874.PubMedCrossRefGoogle Scholar
  33. 33.
    Modjtahedi H, Affleck K, Stubberfield C, Dean C. EGFR blockade by tyrosine kinase inhibitor or monoclonal antibody inhibits growth, directs terminal differentiation and induces apoptosis in the human squamous cell carcinoma HN5. Int J Oncol. 1998;13(2):335–42.PubMedGoogle Scholar
  34. 34.
    de Bono JS, Rowinsky EK. The ErbB receptor family: a therapeutic target for cancer. Trends Mol Med. 2002;8(4 Suppl):S19–26.PubMedCrossRefGoogle Scholar
  35. 35.
    Cunningham D, Humblet Y, Siena S, Khayat D, Bleiberg H, Santoro A, et al. Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. N Engl J Med. 2004;351(4):337–45. doi: 10.1056/NEJMoa033025.PubMedCrossRefGoogle Scholar
  36. 36.
    Baselga J, Arteaga CL. Critical update and emerging trends in epidermal growth factor receptor targeting in cancer. J Clin Oncol. 2005;23(11):2445–59. doi: 10.1200/JCO.2005.11.890.PubMedCrossRefGoogle Scholar
  37. 37.
    Bonner JA, Harari PM, Giralt J, Azarnia N, Shin DM, Cohen RB, et al. Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med. 2006;354(6):567–78. doi: 10.1056/NEJMoa053422.PubMedCrossRefGoogle Scholar
  38. 38.
    Zhu H, Cao X, Ali-Osman F, Keir S, Lo HW. EGFR and EGFRvIII interact with PUMA to inhibit mitochondrial translocalization of PUMA and PUMA-mediated apoptosis independent of EGFR kinase activity. Cancer Lett. 2010;294(1):101–10. doi: 10.1016/j.canlet.2010.01.028.PubMedCrossRefGoogle Scholar
  39. 39.
    Cao X, Zhu H, Ali-Osman F, Lo HW. EGFR and EGFRvIII undergo stress- and EGFR kinase inhibitor-induced mitochondrial translocalization: a potential mechanism of EGFR-driven antagonism of apoptosis. Mol Cancer. 2011;10:26. doi: 10.1186/1476-4598-10-26.PubMedCrossRefGoogle Scholar
  40. 40.
    Yao Y, Wang G, Li Z, Yan B, Guo Y, Jiang X, et al. Mitochondrially localized EGFR is independent of its endocytosis and associates with cell viability. Acta Biochim Biophys Sin (Shanghai). 2010;42(11):763–70. doi: 10.1093/abbs/gmq090.CrossRefGoogle Scholar
  41. 41.
    Cvrljevic AN, Akhavan D, Wu M, Martinello P, Furnari FB, Johnston AJ, et al. Activation of Src induces mitochondrial localisation of de2-7EGFR (EGFRvIII) in glioma cells: implications for glucose metabolism. J Cell Sci. 2011;124(Pt 17):2938–50. doi: 10.1242/jcs.083295.PubMedCrossRefGoogle Scholar
  42. 42.
    Burgess AW, Cho HS, Eigenbrot C, Ferguson KM, Garrett TP, Leahy DJ, et al. An open-and-shut case? Recent insights into the activation of EGF/ErbB receptors. Mol Cell. 2003;12(3):541–52.PubMedCrossRefGoogle Scholar
  43. 43.
    Frederick L, Eley G, Wang XY, James CD. Analysis of genomic rearrangements associated with EGRFvIII expression suggests involvement of Alu repeat elements. Neuro Oncol. 2000;2(3):159–63.PubMedGoogle Scholar
  44. 44.
    Xue L, Lucocq J. ERK2 signalling from internalised epidermal growth factor receptor in broken A431 cells. Cell Signal. 1998;10(5):339–48.PubMedCrossRefGoogle Scholar
  45. 45.
    Sorkin A. Internalization of the epidermal growth factor receptor: role in signalling. Biochem Soc Trans. 2001;29(Pt 4):480–4.PubMedCrossRefGoogle Scholar
  46. 46.
    Levy EM, Sycz G, Arriaga JM, Barrio MM, von Euw EM, Morales SB, et al. Cetuximab-mediated cellular cytotoxicity is inhibited by HLA-E membrane expression in colon cancer cells. Innate Immun. 2009;15(2):91–100. doi: 10.1177/1753425908101404.PubMedCrossRefGoogle Scholar
  47. 47.
    Adams GP, Weiner LM. Monoclonal antibody therapy of cancer. Nat Biotechnol. 2005;23(9):1147–57. doi: 10.1038/nbt1137.PubMedCrossRefGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2011

Authors and Affiliations

  • Agnieszka Dreier
    • 1
  • Stefan Barth
    • 2
  • Anand Goswami
    • 1
  • Joachim Weis
    • 1
  1. 1.Institute of Neuropathology, Medical FacultyRWTH Aachen UniversityAachenGermany
  2. 2.Institute of Biomedical Engineering, Division of Experimental Medicine and Immunotherapy, Medical FacultyRWTH Aachen UniversityAachenGermany

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