Abstract 2491: Beclin1 promotes DNA double-strand breaks repair

Abstract

Abstract Beclin1 is a well-established core mammalian autophagy protein that has been presumed to suppress oncogenesis via an autophagy-mediated mechanism. Since its discovery, studies have also shown that Beclin1 interacts with an increasing number of cofactors. However, the tumor suppressive roles of Beclin1 remain a major research focus, and the exact mechanism by which Beclin1 acts as a tumor suppressor has remained largely obscure. Our confocal analysis showed that in embryonic and neonatal mice, Beclin1 is primarily located in the cytoplasm in hepatocytes, with a very small portion located in the nucleus. When the mice were 15d, roughly half of the total Beclin1 was located in the nucleus. Thereafter, Beclin1 progressively redistributed from cytoplasma to nucleus within a few weeks after birth. This pattern, was similarly sustained not only in hepatocytes but also in other tissues. And we found both 0-50 and 254-278 residues are required for Beclin1 nuclear localization. Furthermore, loss of Beclin1 resulted in reduced DNA damage repair. We found that the beclin1-/- cells showed a marked increased γ-H2AX foci that are considered as a biomarker of DNA DSBs. Moreover, beclin1-/- cells showed approximately about 60-fold and 30-fold reductions in DSB repair by NHEJ and HR pathways. A series of key DSBs repair proteins were significantly reduced in beclin1-/- cells. Interestingly, Beclin1 regulates DSB repair independent of autophagy. Autophagic flux was maintained in the beclin1-/- cells. Perhaps an alternative autophagy may be activated when beclin1 is deleted as ULK1 was upregulated. We further found that Beclin1 promotes DSB repair via a direct interaction with DNA topoisomerase IIβ (Top2b). Pull down results showed that Top2b is one of the major Beclin1-interacting proteins in the nucleus. And we found that this interaction was intensified after exposure to IR. Top2b and Beclin 1 were found to be colocalized to the DSB sites, after exposure to IR. Strikingly, depletion of Top2b by gene silencing completely prohibited Beclin1 localized to the DNA break sites. We constructed a beclin1-/- cell line using CRISPR/Cas9 system. The spot counts of protein foci were measured by imaging flow cytometry after exposure to 6Gy IR. HR and NHEJ reporter assays were used to detect the activity of DSBs repair pathways. We pulled down nuclear proteins with ectopically expressed Flag-tagged Beclin1. The identities of the Beclin1-interacting proteins were determined using mass spectrometry. Our work establishes that Beclin1 plays an autophagy-independent role in maintaining genomic integrity by promoting DNA damage repair. The finding challenge the long-standing paradigm of the mechanisms that are involved in the autophagy-mediated tumor suppressive role of Beclin1. This finding suggests that when manipulating cytoplasmic Beclin1 that is involved in the autophagy pathway in order to explore targeted tumor therapies, the non-autophagy cytoprotective role of Beclin 1 in the nucleus should also be considered. Citation Format: Fei Xu, Lili Yan, Jianrong Wang, Jinming Yang. Beclin1 promotes DNA double-strand breaks repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2491. doi:10.1158/1538-7445.AM2017-2491