Abstract：
OBJECTIVE: Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer with limited therapeutic options. KRAS mutations are among the most abundant genetic alterations in iCCA associated with poor clinical outcome and treatment response. Recent findings indicate that Poly(ADP-ribose)polymerase1 (PARP-1) is implicated in KRAS-driven cancers, but its exact role in cholangiocarcinogenesis remains undefined.
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.
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.
摘要：
目的：肝内胆管癌（iCCA）是第二常见的原发性肝癌，治疗选择有限。KRAS突变是iCCA中与不良临床结果和治疗反应相关的最丰富的遗传改变之一。最近的研究结果表明，聚（ADP-核糖）聚合酶1（PARP-1）与KRAS驱动的癌症有关，但其在胆管癌发生中的确切作用尚不明确。
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。
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。
