BRAT1 Impairs DNA Damage Repair in Glioblastoma Cell Lines ^†

Abstract

Glioblastomas (GBMs) are one of the most malignant brain tumors in adults. This is partly due to the potential presence of so-called glioma stem-like cells (GSCs), which are characterized by the expression of stemness markers and a resistance to radio- and chemotherapy. Previous work from us showed that after combination treatment of GSCs with arsenic trioxide and gossypol, the protein BRCA1-associated ATM-activator 1 (BRAT1) was one of the most downregulated proteins. This protein is largely undescribed, but it has been shown to regulate DNA damage signaling through interaction with ATM, BRCA1, and DNA-PKcs in initial stages of DNA damage response. An unpublished analysis of The Cancer Genome Atlas and The Human Protein Atlas databases showed an increased expression of BRAT1 in GBMs compared to healthy tissues and that an increased expression is negatively correlated with patient survival. Due to these findings, our goal is to analyze the radio-sensitizing effect of BRAT1 on FCS-grown (i.e., differentiated) highly radio-resistant GBM cells and GSCs. Here, using stable knockdowns of BRAT1, we show that it is needed for effective DNA repair after irradiation using a γH2AX-foci assay, whereas it is dispensable for cellular proliferation. A cell death analysis using Annexin V/propidium iodide staining revealed a first hint that BRAT1 downregulation sensitizes GBM cells to irradiation. Moreover, through immunofluorescent staining, we showed that BRAT1 is needed for BRCA1 recruitment to DNA damage sites. Future experiments will aim at systematically analyzing the downstream effects of BRAT1 depletion and to determine further interactors. Thus, we hope to gain a deeper understanding of the mechanism of radio-resistance in GSCs, also in order to individually determine the effectiveness of radiotherapy.