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EGFRvIII and DNA Double-Strand Break Repair: A Molecular Mechanism for Radioresistance in Glioblastoma

Departments of ¹ Radiation Oncology, ² Internal Medicine, ³ Neurology, ⁴ Pathology, ⁵ Neurological Surgery, and ⁶ Pharmacology, ⁷ Annette G. Strauss Center for Neuro-Oncology, and ⁸ Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas; and ⁹ Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla, California

Requests for reprints: Sandeep Burma, Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, 2201 Inwood Road, NC7.214E, Dallas, TX 75390. Phone: 214-648-7440; Fax: 214-648-5995; E-mail: Sandeep.Burma{at}UTSouthwestern.edu, or Robert M. Bachoo, Department of Neurology, University of Texas Southwestern Medical Center, 2201 Inwood Road, ND3.300, Dallas, TX 75390. Phone: 214-645-6309; Fax: 214-645-6239; E-mail: Robert.Bachoo{at}UTSouthwestern.edu.

Key Words: glioblastoma multiforme (GBM) • radioresistance • DNA double-strand break (DSB) • DNA repair • epidermal growth factor receptor (EGFR) • DNA-dependent protein kinase (DNA-PK)

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. [Cancer Res 2009;69(10):4252–9]