Departments of Pharmacology and Radiation Oncology, Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas.
Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, Texas.
Clin Cancer Res. 2018 Dec 15;24(24):6459-6470. doi: 10.1158/1078-0432.CCR-17-1118. Epub 2018 Aug 14.
Identification of novel strategies to expand the use of PARP inhibitors beyond BRCA deficiency is of great interest in personalized medicine. Here, we investigated the unannotated role of Kub5-Hera (K-H) in homologous recombination (HR) repair and its potential clinical significance in targeted cancer therapy.
Functional characterization of K-H alterations on HR repair of double-strand breaks (DSB) were assessed by targeted gene silencing, plasmid reporter assays, immunofluorescence, and Western blots. Cell survival with PARP inhibitors was evaluated through colony-forming assays and statistically analyzed for correlation with K-H expression in various nonmutated breast cancers. Gene expression microarray/qPCR analyses, chromatin immunoprecipitation, and rescue experiments were used to investigate molecular mechanisms of action.
K-H expression loss correlates with rucaparib LD values in a panel of nonmutated breast cancers. Mechanistically, K-H depletion promotes , where extensive upregulation of PARP1 activity was required for the survival of breast cancer cells. PARP inhibition in these cells led to synthetic lethality that was rescued by wild-type K-H reexpression, but not by a mutant K-H (p.R106A) that weakly binds RNAPII. K-H mediates HR by facilitating recruitment of RNAPII to the promoter region of a critical DNA damage response and repair effector, cyclin-dependent kinase 1 ().
Cancer cells with low K-H expression may have exploitable properties that greatly expand the use of PARP inhibitors beyond BRCA mutations. Our results suggest that aberrant K-H alterations may have vital translational implications in cellular responses/survival to DNA damage, carcinogenesis, and personalized medicine.
在个性化医疗中，寻找新的策略以扩大聚腺苷二磷酸核糖聚合酶（PARP）抑制剂的应用范围，超越 BRCA 缺陷，这是非常有意义的。在这里，我们研究了 KUB5-Hera（K-H）在同源重组（HR）修复中的未被注释的作用，及其在靶向癌症治疗中的潜在临床意义。
通过靶向基因沉默、质粒报告基因检测、免疫荧光和 Western blot 来评估 K-H 对双链断裂（DSB）HR 修复的改变。通过集落形成实验评估 PARP 抑制剂的细胞存活情况，并对其与各种非突变乳腺癌中 K-H 表达的相关性进行统计学分析。采用基因表达微阵列/qPCR 分析、染色质免疫沉淀和挽救实验来研究作用机制。
K-H 表达缺失与一组非突变乳腺癌中鲁卡帕利的 LD 值相关。从机制上讲，K-H 耗竭促进了 PARP1 活性的广泛上调，这是乳腺癌细胞存活所必需的。在这些细胞中，PARP 抑制导致合成致死，野生型 K-H 重新表达可挽救，但突变型 K-H（p.R106A）则不能挽救，因为后者与 RNA 聚合酶 II 结合较弱。K-H 通过促进 RNA 聚合酶 II 募集到关键 DNA 损伤反应和修复效应物细胞周期蛋白依赖性激酶 1（）的启动子区域来介导 HR。
低 K-H 表达的癌细胞可能具有可利用的特性，可极大地扩大 PARP 抑制剂在 BRCA 突变之外的应用范围。我们的结果表明，异常的 K-H 改变可能对细胞对 DNA 损伤、癌变和个性化医疗的反应/存活具有重要的转化意义。
