Impact of Poly(ADP-ribose) polymerase 1 on phenotypic and molecular features in KRAS-driven intrahepatic cholangiocarcinoma

Abstract

Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer with an increasing incidence over recent decades. Due to the complexe pathogenesis and pronounced genetic heterogeneity of iCCA, treatment options are still limited, and the prognosis of patients remains dismal. Activating KRAS mutations are observed in around 12-16% of iCCA patients and characterized by poor response to chemotherapy and reduced overall survival, highlighting the need for novel therapeutic approaches in this subgroup. Several findings indicate that expression of DNA damage response protein Poly (ADP-ribose) polymerase 1 (PARP-1) is preferentially enhanced in KRAS-mutant tumors. However, the exact role of PARP-1 in cholangio-carcinogenesis is poorly understood. In this study, we investigated the impact of PARP-1 in KRAS-mutant iCCA in vitro and in vivo. PARP-1 depletion was generated by siRNA-mediated knockdown and CRISPR/Cas9-mediated knockout and impact on proliferation in vitro was analyzed by viability assay as well as colony and sphere formation. Further, we examined induction of DNA damage response, apoptosis, and oxidative stress upon PARP-1 knockout and control conditions. To investigate impact of PARP-1 deficiency in KRAS-driven tumor initiation, we combined a PARP-1 knockout mouse model with an inducible KRAS-driven mouse model using hydrodynamic tail vein injection (HDTV). Transcriptome analyses of CRISPR/Cas9 PARP-1 knockout clones and in vivo tumor samples were employed to further investigate molecular mechanisms. siRNA-mediated knockdown of PARP-1, as well as treatment with PARP-1 inhibitor olaparib preferentially impaired cell viability and tumorigenicity in KRAS-mutated cell lines. Further, KRAS-mutated cell lines showed higher induction of DNA double-strand breaks and enhanced ROS levels upon PARP-1 knockout compared to wildtype cells. PARP-1 deficiency further considerably impaired biliary carcinogenesis and induced a shift from dominant iCCA towards HCC phenotype in a KRAS-dependent manner in vivo. Transcriptome analyses of CRISPR/Cas9 PARP-1 knockout clones and in vivo tumor specimens revealed differential expression of DNA damage response pathways as well as other cellular pathways known to be affected by PARP-1, e.g. inflammation, oxidative stress, and cell death signaling. Altogether, this study delineates a preferential sensitivity of KRAS-mutant iCCA towards PARP-1-based interventions. PARP-1 inhibition induces functional impairment of tumorigenic capacity as well as oxidative stress response. Integration of in vivo and in vitro transcriptomic data with different established prognostic subgroups of PLC, demonstrated that KRAS-mutant iCCA switch from poor to good prognosis upon PARP-1 depletion. Taken together, these findings suggest an unrecognized prognostic and therapeutic role of PARP-1 in iCCA patients with oncogenic KRAS signaling.