KRAS-mutated iCCA display distinct molecular alterations and a preferential sensitivity towards PARP-1 inhibition

Introduction Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer with an increasing incidence over recent years. Due to the complexity of iCCA pathogenesis and the pronounced genetic heterogeneity treatment options are still limited. KRAS mutations are observed in a sizeable subgroup of iCCA patients and characterized by poor response to chemotherapy and reduced overall survival, highlighting the need for novel therapeutic approaches. Several findings indicate that DNA damage response protein Poly (ADP-ribose) polymerase 1 (PARP-1) preferentially affects KRAS-mutated iCCA cell lines, but the exact mechanism of PARP-1 in cholangiocarcinogenesis remains elusive.

Material & Methods siRNA-mediated knockdown of PARP-1 and treatment with PARP-1-inhibitor AZD2281 were conducted in KRAS-mutated and non-mutated iCCA cell lines. Functional assessment of PARP-1 knockout and inhibition on tumorigenic potential was analyzed by viability assay and colony and sphere formation. Further, we examined cellular reactive oxygen species (ROS) levels using DCFDA assay. Further, we used a PARP-1 knockout mouse model inducing specific KRAS-mutated tumors by hydrodynamic tail vein injection (HDTV). Transcriptome analyses of CRISPR/Cas9 PARP-1 knockout clones were employed to further investigate underlying molecular mechanisms.

Results siRNA-mediated knockdown of PARP-1, as well as treatment with PARP-1 inhibitor preferentially impaired cell viability and tumorigenicity in KRAS-mutated cell lines and led to a 40-45 % reduction in colony and sphere formation capacity. Further, KRAS-mutated cell lines showed higher basal ROS levels after PARP-1 knockout and higher generation of ROS after induction of oxidative stress via H2O2 treatment. In vivo induced tumors induced by injection of KRAS/p53 showed a distinct phenotype in PARP-1 deficient background. Transcriptome analyses of CRISPR/Cas9 PARP-1 knockout clones revealed differential expression of DNA damage response pathways as well as other cellular pathways known to be affected by PARP-1, e.g. oxidative stress, c-MYC, and cell death signaling.

Conclusion This study confirms a preferential sensitivity of KRAS-mutated iCCA towards PARP-1-based interventions. PARP-1 inhibition induces functional impairment of tumorigenic capacity and induction of oxidative stress. These findings suggest an unrecognized therapeutic role of PARP-1 in this poor prognostic subgroup of iCCA patients.

Publikationsverlauf

Artikel online veröffentlicht:
04. Januar 2021