METHODS: PARP-1 inhibition was performed in patient-derived and established iCCA cells using RNAi, CRISPR/Cas9 and pharmacological inhibition in KRAS-mutant, non-mutant cells. In addition, Parp-1 knockout mice were combined with iCCA induction by hydrodynamic tail vein injection to evaluate an impact on phenotypic and molecular features of Kras-driven and Kras-wildtype iCCA. Clinical implications were confirmed in authentic human iCCA.
RESULTS: PARP-1 was significantly enhanced in KRAS-mutant human iCCA. PARP-1-based interventions preferentially impaired cell viability and tumourigenicity in human KRAS-mutant cell lines. Consistently, loss of Parp-1 provoked distinct phenotype in Kras/Tp53-induced versus Akt/Nicd-induced iCCA and abolished Kras-dependent cholangiocarcinogenesis. Transcriptome analyses confirmed preferential impairment of DNA damage response pathways and replicative stress response mediated by CHK1. Consistently, inhibition of CHK1 effectively reversed PARP-1 mediated effects. Finally, Parp-1 depletion induced molecular switch of KRAS-mutant iCCA recapitulating good prognostic human iCCA patients.
CONCLUSIONS: Our findings identify the novel prognostic and therapeutic role of PARP-1 in iCCA patients with activation of oncogenic KRAS signalling.
方法:使用RNAi在患者来源和建立的iCCA细胞中进行PARP-1抑制,CRISPR/Cas9和KRAS突变体的药理学抑制,非突变细胞。此外,通过流体动力学尾静脉注射将Parp-1敲除小鼠与iCCA诱导组合,以评估对Kras驱动和Kras野生型iCCA的表型和分子特征的影响。在真正的人iCCA中证实了临床意义。
结果:PARP-1在KRAS-突变型人iCCA中显著增强。基于PARP-1的干预措施优先损害人KRAS突变细胞系的细胞活力和致瘤性。始终如一,与Akt/Nicd诱导的iCCA相比,Parp-1的丢失在Kras/Tp53诱导的iCCA中引起了不同的表型,并消除了Kras依赖性的胆管癌发生。转录组分析证实了由CHK1介导的DNA损伤应答途径和复制应激反应的优先损害。始终如一,抑制CHK1可有效逆转PARP-1介导的效应。最后,Parp-1耗竭诱导的KRAS突变iCCA分子开关概述了良好预后的人类iCCA患者。
结论:我们的发现确定了PARP-1在具有致癌KRAS信号激活的iCCA患者中的新的预后和治疗作用。