DataSheet_1_Analysis of Adaptive Olaparib Resistance Effects on Cisplatin Sensitivity in Triple Negative Breast Cancer Cells.docx

Poly-(ADP)-ribose polymerase inhibitors (PARPi) and platinum-based drugs are promising therapies for triple negative breast cancers (TNBC) with BRCA1 or BRCA2 loss. PARPi(s) show better efficacies when combined with platinum-based therapy, however, acquisition of PARPi resistance has been linked with co-resistance to platinum-based drugs. Here, we show that TNBCs with constitutively hyperactivated PARP-1 display greater tolerances for the PARPi olaparib and cisplatin, and respond synergistically to olaparib/cisplatin combinations with increased cytotoxicity. Regardless of BRCA1 and PARP-1 activity status, upon gaining olaparib resistance (OlaR), OlaR MDA-MB-468 (BRCA1 wild-type) and SUM1315 (BRCA1 mutant) TNBC cells retain cisplatin sensitivities of their isogenic parental counterparts. OlaR TNBC cells express decreased levels of PARP-1 and Pol η, a translesion-synthesis polymerase important in platinum-induced interstrand crosslink repair. Although native RAD51 recombinase levels are unaffected, anti-RAD51 immunoreactive low molecular weight sbands are exclusively detected in OlaR cells. Despite normal BRCA1, RAD51 foci formation/recruitment to double-strand breaks are impaired in OlaR MDA-MB-468 cells, suggesting homologous-recombination impairment. RNA-seq and pathway analysis of cisplatin-affected genes revealed enrichment of G2/M cell cycle regulation and DNA repair pathways in parental and OlaR MDA-MB-468 cells whereas parental and OlaR SUM1315 cells showed enrichment of inflammatory stress response pathways associated with TNFR1/2, TWEAK and IL-17 signaling. These data show that TNBC models with wild type versus mutant BRCA1 exhibit differences in CDDP-induced cellular response pathways, however, the CDDP-induced signaling responses remain stable across the isogenic models of OlaR from the same lineage. These data also show that adaptive OlaR does not automatically promote cisplatin resistance, implicating the potential benefit of platinum-based therapy for OlaR TNBCs.

多聚ADP核糖聚合酶抑制剂（Poly-(ADP)-ribose polymerase inhibitors，PARPi）与铂类药物，是携带BRCA1或BRCA2缺失的三阴性乳腺癌（triple negative breast cancers，TNBC）的极具前景的治疗方案。PARPi联合铂类治疗时可展现更优疗效，但PARPi耐药的获得常与铂类药物交叉耐药相关。本研究发现，组成型高度激活PARP-1（PARP-1）的TNBC细胞，对PARPi奥拉帕利（olaparib）和顺铂（cisplatin）的耐受性更强，且奥拉帕利/顺铂联合给药可产生协同细胞毒性作用。无论BRCA1与PARP-1的活性状态如何，获得奥拉帕利耐药（olaparib resistance，OlaR）的OlaR MDA-MB-468（BRCA1野生型）与SUM1315（BRCA1突变型）TNBC细胞，仍保留其同基因亲本细胞对顺铂的敏感性。OlaR TNBC细胞中PARP-1与Pol η（聚合酶η，Pol η）的表达水平降低，Pol η是一种在铂类诱导的链间交联修复中发挥重要作用的跨损伤合成聚合酶。尽管天然RAD51重组酶（RAD51 recombinase）的表达水平未受影响，但仅在OlaR细胞中可检测到抗RAD51免疫反应性的低分子量条带。尽管BRCA1功能正常，但OlaR MDA-MB-468细胞中RAD51焦点的形成与向双链断裂位点的招募过程受损，提示存在同源重组缺陷。对顺铂调控基因的RNA测序（RNA-seq）与通路分析结果显示，亲本与OlaR MDA-MB-468细胞中富集了G2/M细胞周期调控与DNA修复通路；而亲本与OlaR SUM1315细胞则富集了与TNFR1/2、TWEAK及IL-17信号通路相关的炎症应激反应通路。上述数据表明，携带野生型与突变型BRCA1的TNBC模型，其顺铂诱导的细胞反应通路存在差异；但来自同一谱系的OlaR同基因模型中，顺铂诱导的信号通路反应保持稳定。本研究数据同时表明，适应性获得的奥拉帕利耐药并不会自动引发顺铂耐药，这提示铂类治疗对OlaR TNBC患者具有潜在应用价值。