Abstract A24: A cascade of masked nuclear export signals regulates the BRCA2 tumor suppressor pathway

Inherited germline mutations in a single copy of the BRCA2 tumor suppressor predispose individuals to breast, ovarian, pancreatic and other cancers. BRCA2 exerts its tumor suppressive function in the cell nucleus through its role in the repair of DNA breaks by homologous recombination, where it controls the accumulation of the RAD51 recombinase enzyme at sites of DNA breakage. Cancer associated in-frame point mutations in BRCA2 have been shown to result in cytoplasmic localization of the protein, but the mechanism underlying this is unclear. Here, we identify an unrecognized mechanism controlling the nuclear localization of BRCA2 and its cargo RAD51, which is disrupted by the common cancer-associated mis-sense mutation BRCA2 D2723H . A nuclear export signal (NES) in BRCA2 is masked by its interaction with a partner protein, DSS1, such that point mutations impairing BRCA2-DSS1 binding render BRCA2 cytoplasmic. In turn, cytoplasmic mis-localization of mutant BRCA2 inhibits the nuclear retention of RAD51, by exposing a similar NES in RAD51 that is usually obscured by the BRCA2-RAD51 interaction. We demonstrate the functionality of these export sequences by cellular localization assays and in vitro binding analyses to the exportin CRM1. We note two key implications of our work. Firstly, CRM1 mediated nuclear export is relevant to the localization of tumor suppressors such as p53 and BRCA1. However, this is the first report to our knowledge, of a ‘cascade’ of NES-masking interactions mediating nuclear localization for a tumor suppressor pathway. Our findings suggest the exploration of analogous mechanisms in other cancer-relevant cellular networks. Secondly, we also note that BRCA2D2723H decreases RAD51 nuclear retention even when wildtype BRCA2 is present. This is consistent with a transdominant effect for at least a fraction of the heterozygous disease-associated mutations noted in BRCA2 . We find an increase in endogenous DNA damage in cells heterozygous for the D2723H mutation, suggesting that BRCA2 D2723H heterozygosity causes a cumulative effect on genome stability in patients, which acts over years to promote carcinogenesis. Transdominance may also explain the paradox of tumorigenesis occurring in certain cases without a somatic second hit to BRCA2. Together, our results link the nucleo-cytoplasmic translocation of the BRCA2 tumor suppressor to the cellular mechanisms of disease following its germline inactivation. Citation Format: Anand D. Jeyasekharan, Yang Liu, Hiroyoshi Hattori, Venkat Pisupati, Eeson Rajendra, Asta Bjork Jonsdottir, Miyoung Lee, Elayanambi Sundaramoorthy, Simon Schlachter, Ashok Venkitaraman. A cascade of masked nuclear export signals regulates the BRCA2 tumor suppressor pathway. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A24.