Abstract 3944: Caveolin-mediated Tie2 nuclear translocation results in enhanced NHEJ repair and glioma radioresistance
Authors: Hossain Mohammad B. , Cortes-Santiago Nahir , Fan Xuejun , Gabrusiewicz Konrad , Gumin Joy , Sulman Erik P. , Lang Frederick , Sawaya Raymond , Yung W.K.Alfred , Fueyo Juan , Gomez-Manzano Candelaria 
Published: May 2015

Glioblastoma is the most frequent subtype of primary brain tumor in adults and are resistant to current strategies of surgery, irradiation, and chemotherapy. Patients suffering from this disease exhibit a median survival that ranges from 9 to 15 months. Glioblastomas invariably recur after therapy due to the presence of cells exhibiting a multitherapy-resistance phenotype. To decipher the aberrant DNA repair pathways that enable tumor cells to survive DNA damage upon chemotherapy and radiotherapy should guide us to provide our patients with a more efficacious therapy. The abnormal function of tyrosine kinase receptors (TKRs) is a hallmark of malignant gliomas. Our group reported the expression of the TKR Tie2 in in brain tumor stem cells (BTSCs) and in human surgical glioma specimens in relation to malignancy. In in vivo experiments, consisting of ionizing irradiation (IR) of mice bearing intracranial BTSCs-derived xenografts showed unexpected Tie2 nuclear localization. We observed that Tie2 traffics from the cellular membrane to the nucleus upon IR stimuli, as assessed by immunoflurorescence studies using confocal microscope and subcellular fractionation followed by Western blots. This phenomenon is ligand dependent, as increased levels of Tie2 natural ligand, Angiopoietin1 (Ang1), were observed after ionizing radiation, and nuclear Tie2 levels were decreased using soluble Tie2, which blocks the Ang1/Tie2 interaction. Of clinical significance, the presence of Tie2 in the nucleus is significantly associated with radioresistance, as observed by cell viability and clonogenic assays. Tie2 trafficking was associated to Caveolin-1, and this functional complex was disturbed when Caveolin-1 inhibitor or siCaveolin-1 were used, resulting in decreased Tie2 nuclear levels. In addition, we observed that upon IR treatment, nuclear Tie2 bound to DNA/protein complexes, and specifically to the key DNA repair protein γH2AX, as a component of the DNA-repair foci. Based on these results, we hypothesized that Tie2 was involved in DNA damage repair and focused on one of the main pathways involved in double strand break (IR induced), the non-homologous end-joining (NHEJ) repair mechanism. By using a fluorescent reporter construct in which a functional GFP gene is reconstituted following an NHEJ event (gift from Dr. Gorbunova, University of Rochester), we observed that Tie2-expressing cells displayed a more efficient NHEJ repair than Tie2 negative counterparts. Mutation of a Tie2 nuclear localization signal significantly decreased NHEJ efficiency repair and radiosensitization of BTSCs, suggesting a role of nuclear Tie2 in DNA damage repair, specifically in the NHEJ repair mechanism. Collectively, our results should propel the development of preclinical studies of the combination of nuclear Tie2-targeting strategies with radiotherapy for patients with glioblastomas.

Citation Format: Mohammad B. Hossain, Nahir Cortes-Santiago, Xuejun Fan, Konrad Gabrusiewicz, Joy Gumin, Erik P. Sulman, Frederick Lang, Raymond Sawaya, W.K.Alfred Yung, Juan Fueyo, Candelaria Gomez-Manzano. Caveolin-mediated Tie2 nuclear translocation results in enhanced NHEJ repair and glioma radioresistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3944. doi:10.1158/1538-7445.AM2014-3944