Abstract
About 30% of triple negative breast cancers (TNBC) are BRCA defective and show Homologous Recombination DNA repair Deficiency (HRD) and increased sensitivity to genotoxic drugs. We hypothesized that BRCA-defective TNBC are highly sensitive to replicative stress inducing drugs, which could open therapeutic perspectives. Our results show that BRCA1-deficient (BRCA1-Def) TNBC, as well as BRCA1-Def ovarian cancer cell lines showed increased sensitivity to Gemcitabine. Noticeably, Gemcitabine induced increased cell death in BRCA1-Def cells, associated with mediocre replicative stress management. Indeed, up to 80% of BRCA1-Def cells displayed persistent gH2AX staining even 48h after washing off Gemcitabine, compared with their BRCA1-WT counterpart in which staining had decreased significantly. Furthermore, Gemcitabine treated BRCA1-Def cells showed a persistent imbalance between RPA32-positive and gH2Ax-positive cells, suggesting unresolved replication stress in these cells and occurrence of replication catastrophe in a substantial fraction of the cells. Furthermore, an important fraction of gH2AX+ cells displayed pan-nuclear staining, which numbers steadily increased over time in Gemcitabine treated BRCA1-Def cells, while they decreased in the BRCA1-WT counterpart. Interestingly, nearly 90% of gH2AX pan-nuclear cells were completely negative for RPA32 and showed a strong BrdU staining in non-denaturing conditions, indicating an important accumulation of single stranded DNA specifically in these cells. Noticeably, an important number of cells with pan-nuclear gH2Ax staining were also negative for RAD51 foci, but were positive for both 53BP1 and FANCD2 foci. The opposite landscape was observed in the BRCA1-WT cells in which gH2AX pan nuclear cells were RAD51+ and 53BP1- and FANCD2-. These results indicate an acute replicative stress response in the BRCA1-Def context that induces replication forks arrest. This could lead to replication fork collapse and possibly massive DNA Double Strand Breaks (DSB). To test this hypothesis, we performed Pulse Field Gel Electrophoresis experiments that clearly showed important accumulation of DSBs in the BRCA1-Def cells 48h after Gemcitabine release. These DSBs did not result from the apoptotic cleavage of DNA as joint treatment with Gemcitabine and the apoptosis inhibitor Z-VAD FMK did not modify the DSB level, suggesting that DSBs resulted from an accumulation of unprotected ssDNA. Next we tested whether the accumulation of ssDNA resulted from over-resection caused by uncontrolled activity of the exonuclease MRE11 in BRCA-Def cell and applied a joint treatment of Gemcitabine and Mirin. As a matter of fact, inhibition of MRE11 with Mirin resulted in a specific decrease of the number of cells presenting the pan-nuclear gH2AX staining. Finally, we noted that BRCA1-Def cells were prone to mitotic slippage leading to mitotic catastrophes illustrated by an important accumulation of micronuclei in these cells. Noticeably, micro-nuclei showed double positive BrdU and pan nuclear gH2AX staining indicating that they corresponded to fragmentation of nuclei with elevated ssDNA content. We have also been able to show that the hypersensitivity of BRCA1-Def cancers to acute replication stress is reproducible in vivo in PDX models, which displayed a pan-nuclear gH2AX staining profiles, in accordance with their Gemcitabine response status. Hence, we propose that pan-nuclear gH2Ax staining could be a marker of replication catastrophe in treated tumors and could possibly be considered as a biomarker of the response to replicative stress.