Topoisomerase I Inhibition Radiosensitizing Hepatocellular Carcinoma by RNF144A-mediated DNA-PKcs Ubiquitination and Natural Killer Cell Cytotoxicity

Background and Aims: Topoisomerase I (TOP1) participates the repair of DNA double-strand breaks (DSBs) upon radiation therapy (RT). RNF144A mediates ubiquitination of catalytic subunit of DNA protein kinase (DNA-PKcs), a critical factor in DSB repair. This study aimed to investigate the natural killer (NK) cell-mediated radiosensitization with TOP1 inhibition and the mechanism by DNA-PKcs/RNF144A.Methods: In vitro synergism with TOP1i or cocultured NK cells and RT were evaluated in human hepatocellular carcinoma (HCC) cell lines (Huh7/PLC5) by clonogenic survivals. Orthotopic xenografts were treated with Lipotecan and/ or RT. Protein expression was analyzed by western blotting, immunoprecipitation, subcellular fractionation, and confocal microscopy.Results: Lipotecan/RT had a superior synergistic effect to RT on HCC cells. Combined RT/Lipotecan reduced the xenograft size by 7-fold than RT (p<0.05). Lipotecan caused more radiation-induced DNA damage and DNA-PKcs signaling. The expression of major histocompatibility complex class I-related chain A and B (MICA/B) on tumor cells is associated with the sensitivity to NK cell-mediated lysis. Cocultured NK and HCC cells with Lipotecan radiosensitized HCC cells/tissues with the expression of MICA/B. RNF144A increased more in Huh7 cells with combined RT/TOP1i, and reduced the prosurvival function of DNA-PKcs. The effect was reversed by inhibiting the ubiquitin/proteasome system. In comparison, RNF144A decreased through nuclear translocation with the cumulated DNA-PKcs and radio-resistance of PLC5 cells.Conclusions: TOP1i reinforces NK cell-activated anti-HCC effect of RT through RNF144A mediated DNA-PKcs ubiquitination. RNF144A provides a reason for differentiating radiosensitization effect between HCC cells.