EGFRvIII and DNA double-strand break repair: a molecular mechanism for radioresistance in glioblastoma.

Glioblastoma multiforme (GBM) is the most lethal of brain tumors and is highly resistant to ionizing radiation (IR) and chemotherapy. Here, we report on a molecular mechanism by which a key glioma-specific mutation, epidermal growth factor receptor variant III (EGFRvIII), confers radiation resistance. Using Ink4a/Arf-deficient primary mouse astrocytes, primary astrocytes immortalized by p53/Rb suppression, as well as human U87 glioma cells, we show that EGFRvIII expression enhances clonogenic survival following IR. This enhanced radioresistance is due to accelerated repair of DNA double-strand breaks (DSB), the most lethal lesion inflicted by IR. The EGFR inhibitor gefitinib (Iressa) and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 attenuate the rate of DSB repair. Importantly, expression of constitutively active, myristylated Akt-1 accelerates repair, implicating the PI3K/Akt-1 pathway in radioresistance. Most notably, EGFRvIII-expressing U87 glioma cells show elevated activation of a key DSB repair enzyme, DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Enhanced radioresistance is abrogated by the DNA-PKcs-specific inhibitor NU7026, and EGFRvIII fails to confer radioresistance in DNA-PKcs-deficient cells. In vivo, orthotopic U87-EGFRvIII-derived tumors display faster rates of DSB repair following whole-brain radiotherapy compared with U87-derived tumors. Consequently, EGFRvIII-expressing tumors are radioresistant and continue to grow following whole-brain radiotherapy with little effect on overall survival. These in vitro and in vivo data support our hypothesis that EGFRvIII expression promotes DNA-PKcs activation and DSB repair, perhaps as a consequence of hyperactivated PI3K/Akt-1 signaling. Taken together, our results raise the possibility that EGFR and/or DNA-PKcs inhibition concurrent with radiation may be an effective therapeutic strategy for radiosensitizing high-grade gliomas.

[1]  Felix Y. Feng,et al.  Integration of EGFR inhibitors with radiochemotherapy , 2006, Nature Reviews Cancer.

[2]  P. Harari,et al.  Modulation of radiation response after epidermal growth factor receptor blockade in squamous cell carcinomas: inhibition of damage repair, cell cycle kinetics, and tumor angiogenesis. , 2000, Clinical Cancer Research.

[3]  David E Levy,et al.  Identification of a PTEN-regulated STAT3 brain tumor suppressor pathway. , 2008, Genes & development.

[4]  Adam Dicker,et al.  The contribution of epidermal growth factor receptor (EGFR) signaling pathway to radioresistance in human gliomas: a review of preclinical and correlative clinical data. , 2004, International journal of radiation oncology, biology, physics.

[5]  R. Schmidt-Ullrich,et al.  Radiation-induced autophosphorylation of epidermal growth factor receptor in human malignant mammary and squamous epithelial cells. , 1996, Radiation research.

[6]  N. Curtin,et al.  Radiosensitization and DNA repair inhibition by the combined use of novel inhibitors of DNA-dependent protein kinase and poly(ADP-ribose) polymerase-1. , 2003, Cancer research.

[7]  A. Maity,et al.  Inhibition of Phosphatidylinositol-3-OH Kinase/Akt Signaling Impairs DNA Repair in Glioblastoma Cells following Ionizing Radiation* , 2007, Journal of Biological Chemistry.

[8]  Mitsuo Sato,et al.  Somatic mutations in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) abrogate EGFR-mediated radioprotection in non-small cell lung carcinoma. , 2007, Cancer research.

[9]  Shaomeng Wang,et al.  Targeting of AKT1 enhances radiation toxicity of human tumor cells by inhibiting DNA-PKcs-dependent DNA double-strand break repair , 2008, Molecular Cancer Therapeutics.

[10]  J. Contessa,et al.  EGFRvIII-mediated radioresistance through a strong cytoprotective response , 2003, Oncogene.

[11]  A. Ashworth,et al.  Interaction of the epidermal growth factor receptor and the DNA-dependent protein kinase pathway following gefitinib treatment , 2006, Molecular Cancer Therapeutics.

[12]  M. Berger,et al.  Epidermal growth factor receptor, protein kinase B/Akt, and glioma response to erlotinib. , 2005, Journal of the National Cancer Institute.

[13]  D. Housman,et al.  Oncogenic EGFR signaling cooperates with loss of tumor suppressor gene functions in gliomagenesis , 2009, Proceedings of the National Academy of Sciences.

[14]  O. Bogler,et al.  A common mutant epidermal growth factor receptor confers enhanced tumorigenicity on human glioblastoma cells by increasing proliferation and reducing apoptosis. , 1996, Cancer research.

[15]  Caterina Giannini,et al.  Immunohistochemical Detection of EGFRvIII in High Malignancy Grade Astrocytomas and Evaluation of Prognostic Significance , 2004, Journal of neuropathology and experimental neurology.

[16]  J. Uhm.  Comprehensive genomic characterization defines human glioblastoma genes and core pathways , 2009 .

[17]  E. Glatstein.  Non–Small Cell Lung Cancers with Kinase Domain Mutations in the Epidermal Growth Factor Receptor Are Sensitive to Ionizing Radiation , 2008 .

[18]  Shaomeng Wang,et al.  Blockage of Epidermal Growth Factor Receptor-Phosphatidylinositol 3-Kinase-AKT Signaling Increases Radiosensitivity of K-RAS Mutated Human Tumor Cells In vitro by Affecting DNA Repair , 2006, Clinical Cancer Research.

[19]  Thomas Helleday,et al.  DNA repair pathways as targets for cancer therapy , 2008, Nature Reviews Cancer.

[20]  C. Cordon-Cardo,et al.  Catalytic subunit of DNA-dependent protein kinase: impact on lymphocyte development and tumorigenesis. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[21]  C. Blattner,et al.  p53 stabilization in response to DNA damage requires Akt/PKB and DNA-PK , 2008, Proceedings of the National Academy of Sciences.

[22]  J. Contessa,et al.  Ionizing radiation activates Erb-B receptor dependent Akt and p70 S6 kinase signaling in carcinoma cells , 2002, Oncogene.

[23]  D. Hochhauser,et al.  Modulation of DNA Repair In vitro after Treatment with Chemotherapeutic Agents by the Epidermal Growth Factor Receptor Inhibitor Gefitinib (ZD1839) , 2004, Clinical Cancer Research.

[24]  Erez M. Bublil,et al.  The EGF receptor family : spearheading a merger of signaling and therapeutics , 2007 .

[25]  B. Hemmings,et al.  PKBalpha/Akt1 acts downstream of DNA-PK in the DNA double-strand break response and promotes survival. , 2008, Molecular cell.

[26]  Jesse D. Martinez,et al.  Time and dose-dependent radiosensitization of the glioblastoma multiforme U251 cells by the EGF receptor tyrosine kinase inhibitor ZD1839 ('Iressa'). , 2003, Cancer letters.

[27]  B. Kavanagh,et al.  Radiation-induced proliferation of the human A431 squamous carcinoma cells is dependent on EGFR tyrosine phosphorylation , 1997, Oncogene.

[28]  J. Uhm.  An Integrated Genomic Analysis of Human Glioblastoma Multiforme , 2009 .

[29]  David J. Chen,et al.  DNA-dependent Protein Kinase-independent Activation of p53 in Response to DNA Damage* , 1999, The Journal of Biological Chemistry.

[30]  L. Chin,et al.  Malignant astrocytic glioma: genetics, biology, and paths to treatment. , 2007, Genes & development.

[31]  R. DePinho,et al.  Epidermal growth factor receptor and Ink4a/Arf: convergent mechanisms governing terminal differentiation and transformation along the neural stem cell to astrocyte axis. , 2002, Cancer cell.

[32]  David J. Chen,et al.  Radiation-induced Epidermal Growth Factor Receptor Nuclear Import Is Linked to Activation of DNA-dependent Protein Kinase* , 2005, Journal of Biological Chemistry.

[33]  R. Schmidt-Ullrich,et al.  Radiation-induced release of transforming growth factor alpha activates the epidermal growth factor receptor and mitogen-activated protein kinase pathway in carcinoma cells, leading to increased proliferation and protection from radiation-induced cell death. , 1999, Molecular biology of the cell.

[34]  S. Baker.  PTEN Enters the Nuclear Age , 2007, Cell.

[35]  S. Lees-Miller.  PIKK-ing a new partner: a new role for PKB in the DNA damage response. , 2008, Cancer cell.

[36]  Jack Miller,et al.  Modulation of the DNA-damage response to HZE particles by shielding. , 2008, DNA repair.

[37]  David J. Chen,et al.  Cell Cycle Dependence of DNA-dependent Protein Kinase Phosphorylation in Response to DNA Double Strand Breaks* , 2005, Journal of Biological Chemistry.

[38]  N. Hynes,et al.  Amplification and differential expression of members of theerbB-gene family in human glioblastoma , 2005, Journal of Neuro-Oncology.

[39]  D. Alessi,et al.  Use of kinase inhibitors to dissect signaling pathways. , 2000, Methods in molecular biology.

[40]  David J. Chen,et al.  Role of non-homologous end joining (NHEJ) in maintaining genomic integrity. , 2006, DNA repair.

[41]  A. Friedman,et al.  Resistance to Tyrosine Kinase Inhibition by Mutant Epidermal Growth Factor Receptor Variant III Contributes to the Neoplastic Phenotype of Glioblastoma Multiforme , 2004, Clinical Cancer Research.

[42]  A. Scott,et al.  Monoclonal antibody 806 inhibits the growth of tumor xenografts expressing either the de2-7 or amplified epidermal growth factor receptor (EGFR) but not wild-type EGFR. , 2001, Cancer research.

[43]  David J. Chen,et al.  Role of DNA-PK in the cellular response to DNA double-strand breaks. , 2004, DNA repair.

[44]  H. Wiley,et al.  The Enhanced Tumorigenic Activity of a Mutant Epidermal Growth Factor Receptor Common in Human Cancers Is Mediated by Threshold Levels of Constitutive Tyrosine Phosphorylation and Unattenuated Signaling* , 1997, The Journal of Biological Chemistry.

[45]  Amy J. Hawkins,et al.  Pro-survival AKT and ERK signaling from EGFR and mutant EGFRvIII enhances DNA double-strand break repair in human glioma cells , 2009, Cancer biology & therapy.

[46]  David J. Chen,et al.  DNA-PK phosphorylates histone H2AX during apoptotic DNA fragmentation in mammalian cells. , 2006, DNA repair.

[47]  R. McLendon,et al.  Second messenger systems in human gliomas. , 2007, Archives of pathology & laboratory medicine.

[48]  N. Sergina,et al.  The HER family and cancer: emerging molecular mechanisms and therapeutic targets. , 2007, Trends in molecular medicine.

[49]  Koji Yoshimoto,et al.  Molecular determinants of the response of glioblastomas to EGFR kinase inhibitors. , 2005, The New England journal of medicine.

[50]  W. Cavenee,et al.  A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[51]  R. Schmidt-Ullrich,et al.  Radiosensitization of malignant glioma cells through overexpression of dominant-negative epidermal growth factor receptor. , 2001, Clinical cancer research : an official journal of the American Association for Cancer Research.

[52]  E. Bernhard,et al.  Mutant epidermal growth factor receptor displays increased signaling through the phosphatidylinositol-3 kinase/AKT pathway and promotes radioresistance in cells of astrocytic origin , 2004, Oncogene.

[53]  Reto Meuli,et al.  Promising survival for patients with newly diagnosed glioblastoma multiforme treated with concomitant radiation plus temozolomide followed by adjuvant temozolomide. , 2002, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.