Ovarian Cancers Harbor Defects in Nonhomologous End Joining Resulting in Resistance to Rucaparib.
Ovarian Cancers Harbor Defects in Nonhomologous End Joining Resulting in Resistance to Rucaparib.
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DOI：
10.1158/1078-0432.ccr-16-0564
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发表时间：
2017-04-15
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影响因子：
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通讯作者：
Edmondson RJ
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作者：
McCormick A;Donoghue P;Dixon M;O'Sullivan R;O'Donnell RL;Murray J;Kaufmann A;Curtin NJ;Edmondson RJ
DNA 损伤缺陷在卵巢癌中很常见，可用于分层治疗。尽管大多数工作都集中在同源重组 (HR) 上，但 DNA 双链断裂主要通过非同源末端连接 (NHEJ) 来修复。 NHEJ 缺陷已被证明会导致基因组不稳定，并与化疗耐药性的发展有关。通过测量细胞提取物将线性化质粒单体末端连接成多聚体的能力，在一组卵巢癌细胞系和 47 个原发性腹水衍生卵巢癌培养物中评估 NHEJ。使用 RT-qPCR 和蛋白质印迹法测定 NHEJ 成分的 mRNA 和蛋白表达。使用磺胺罗丹明 B (SRB) 测定评估顺铂和聚（ADP-核糖）聚合酶-1 (PARP) 抑制剂 rucaparib 的细胞毒性。使用 γH2AX/RAD51 病灶测定评估 HR 功能。 NHEJ 在 6 个细胞系中的 4 个和 47 个原代培养物中的 20 个中存在缺陷 (D)。 NHEJ 功能独立于同源重组 (HR) 能力 (C)。 NHEJD 培养物对 rucaparib 具有耐药性 (p=0.0022)。当考虑 HR 和 NHEJ 功能时，仅 NHEJC/HRD 培养物对 rucaparib 敏感（与 NHEJC/HRC p=0.034、NHEJD/HRC p=0.0002 和 NHEJD/HRD p=0.0045 相比）。 DNA-PK 抑制剂 NU7441 诱导 BRCA1 缺陷细胞系对 rucaparib 产生耐药性 (p=0.014) 并恢复 HR 功能。这项研究表明，40% 的卵巢癌中 NHEJ 有缺陷，这与 HR 功能无关，并且与离体原代培养物对 PARP 抑制剂的耐药性相关。
DNA damage defects are common in ovarian cancer and can be used to stratify treatment. Although most work has focussed on Homologous Recombination (HR), DNA double strand breaks are repaired primarily by non-homologous end joining (NHEJ). Defects in NHEJ have been shown to contribute to genomic instability and have been associated with the development of chemoresistance. NHEJ was assessed in a panel of ovarian cancer cell lines and 47 primary ascitic derived ovarian cancer cultures, by measuring the ability of cell extracts to end-join linearized plasmid monomers into multimers. mRNA and protein expression of components of NHEJ was determined using RT-qPCR and western blotting. Cytotoxicities of cisplatin and the Poly(ADP-ribose) polymerase-1 (PARP) inhibitor rucaparib were assessed using sulforhodamine B (SRB) assays. HR function was assessed using γH2AX/RAD51 foci assay. NHEJ was defective (D) in 4 of 6 cell lines and 20 of 47 primary cultures. NHEJ function was independent of homologous recombination (HR) competence (C). NHEJD cultures were resistant to rucaparib (p=0.0022). When HR and NHEJ functions were taken into account, only NHEJC/HRD cultures were sensitive to rucaparib (compared to NHEJC/HRC p=0.034, NHEJD/HRC p=0.0002, and NHEJD/HRD p=0.0045). The DNA-PK inhibitor, NU7441 induced resistance to rucaparib (p=0.014) and HR function recovery in a BRCA1 defective cell line. This study has shown that NHEJ is defective in 40% of ovarian cancers, which is independent of HR function and associated with resistance to PARP inhibitors in ex vivo primary cultures.