PARP inhibition causes premature loss of cohesion in cancer cells

Abstract

Poly(ADP-ribose) polymerases (PARPs) regulate various aspects of cellular function including mitotic progression. Although PARP inhibitors have been undergoing various clinical trials and the PARP1/2 inhibitor olaparib was approved as monotherapy for BRCA-mutated ovarian cancer, their mode of action in killing tumour cells is not fully understood. We investigated the effect of PARP inhibition on mitosis in cancerous (cervical, ovary, breast and osteosarcoma) and noncancerous cells by live-cell imaging. The clinically relevant inhibitor olaparib induced strong perturbations in mitosis, including problems with chromosome alignment at the metaphase plate, anaphase delay, and premature loss of cohesion (cohesion fatigue) after a prolonged metaphase arrest, resulting in sister chromatid scattering. PARP1 and PARP2 depletion suppressed the phenotype while PARP2 overexpression enhanced it, suggesting that olaparib-bound PARP1 and PARP2 rather than the lack of catalytic activity causes this phenotype. Olaparib-induced mitotic chromatid scattering was observed in various cancer cell lines with increased protein levels of PARP1 and PARP2, but not in non-cancer or cancer cell lines that expressed lower levels of PARP1 or PARP2. Interestingly, the sister chromatid scattering phenotype occurred only when olaparib was added during the S-phase preceding mitosis, suggesting that PARP1 and PARP2 entrapment at replication forks impairs sister chromatid cohesion. Clinically relevant DNA-damaging agents that impair replication progression such as topoisomerase inhibitors and cisplatin were also found to induce sister chromatid scattering and metaphase plate alignment problems, suggesting that these mitotic phenotypes are a common outcome of replication perturbation. topoisomerase inhibitors (camptothecin, etoposide) and cisplatin, suggesting that death by mitotic failure is a general phenomenon of perturbed replication. RESULTS Olaparib causes anaphase delay and chromatid scattering in metaphase-arrested cellsIn order to investigate the effect of PARP inhibition on mitosis, we performed live-cell imaging of HeLa cells stably expressing H2B-mCherry together with securin-EGFP [29] treated with olaparib (AZD2281, Ku-0059436) [30], talazoparib (BMN 673) [31] or veliparib (ABT-888) [32] as PARP1/2 inhibitors, XAV-939 as tankyrase1/2 (PARP5a/b) inhibitor [33] and ME328 as PARP3 inhibitor ( Figure 1A) [34]. Of the five tested inhibitors applied at different concentrations for 30 h, only PARP1/2 inhibitors caused anaphase delay measured as the time required for the cells to progress from nuclear envelope breakdown (NEBD) to anaphase (Figure 1A,B). The median NEBD-anaphase duration was extended from 42 min in DMSO-treated control cells to 57 min for 10 μ M olaparibtreated cells, to 60 min for 30 μ M veliparib and to 60 min for 100 nM talazoparib (Figure 1A,B). 40-50% of inhibitor-treated mitotic cells failed to enter anaphase due to metaphase plate formation problems or chromatid scattering after correct metaphase plate formation ( Figure 1B...