Replication-Poison Treatment in BRCA1-Deficient Breast Cancer Causes MRE11 Over-Resection That Induces Single-Stranded DNA Accumulation and Mitotic Catastrophe

Abstract

BRCA1 deficiency is observed in approximately 25% of triple-negative breast cancer (TNBC). BRCA1, a key player of homologous recombination repair, is also involved in stalled DNA replication fork protection and repair. Here, we investigated the sensitivity of BRCA1-deficient TNBC models to the frequently used replication chain terminator gemcitabine, which does not directly induce DNA breaks. A large fraction of BRCA1-deficient cells was sensitive to gemcitabine, in contrast to their isogenic BRCA1-proficient counterparts. Gemcitabine-treated BRCA1-deficient cells accumulated massive levels of ssDNA and presented no RPA or RAD51 nuclear foci. The gemcitabine-induced accumulation of ssDNA in BRCA1-deficient cells was strongly diminished by targeting MRE11 with inhibitors and by siRNA attenuation. In contrast, treatment with the PARP1/2 inhibitor olaparib did not result in MRE11-dependent over-resection. Furthermore, a fraction of gemcitabine-treated BRCA1-deficient cells that showed massive ssDNA accumulation slipped into mitosis, producing mitotic bridges and strongly stained bromodeoxyuridine (BrdU) and γH2AX micronuclei. The BrdU-positive micronuclei and DNA bridges also stained positively for cyclic GMP-AMP synthase. In conclusion, these data suggest that gemcitabine treatment in BRCA1-deficient TNBC exposes unprotected nascent DNA linked to replication fork reversal, which leads to MRE11 over-resection and ssDNA accumulation. Therefore, the observed hypersensitivity to gemcitabine indicates that it could be a beneficial addition to BRCA1-deficient TNBC treatment. Significance: Treating BRCA1-deficient triple-negative breast cancer with gemcitabine induces massive ssDNA production due to MRE11 over-resection and causes cells to slip into mitosis, produce micronuclei, accumulate DNA breaks, and ultimately die.