Genome-wide RNAi screening identifies RFC4 as a factor that mediates radioresistance in colorectal cancer by facilitating non-homologous end joining repair

PURPOSE: Neoadjuvant chemoradiotherapy (neoCRT) is a standard treatment for locally advanced rectal cancer (LARC); however, resistance to chemoradiotherapy (CRT) is one of the main obstacles to improving treatment outcomes. The goal of this study was to identify factors involved in the radioresistance of CRC and to clarify the underlying mechanisms. EXPERIMENTAL DESIGN: A genome-wide RNAi screen was employed to search for candidate radioresistance genes. After RFC4 knockdown or overexpression, CRC cells exposed to X-rays both in vitro and in a mouse model were assayed for DNA damage, cytotoxicity and apoptosis. Moreover, the regulatory effects and mechanisms of RFC4 in DNA repair were investigated in vitro . Finally, the relationships between RFC4 expression and clinical parameters and outcomes were investigated in 145 LARC patients receiving neoCRT. RESULTS: RFC4, NCAPH, SYNE3, LDLRAD2, NHP2, and FICD were identified as potential candidate radioresistance genes. RFC4 protected CRC cells from X-ray-induced DNA damage and apoptosis in vitro and in vivo . Mechanistically, RFC4 promoted non-homologous end joining (NHEJ)-mediated DNA repair by interacting with Ku70/Ku80 but did not affect homologous recombination-mediated repair. Higher RFC4 expression in cancer tissue was associated with weaker tumor regression and poorer prognosis in LARC patients treated with neoCRT, which likely resulted from the effect of RFC4 on radioresistance, not chemoresistance. CONCLUSIONS: RFC4 was identified as a radioresistance factor that promotes NHEJ-mediated DNA repair in CRC cells. Additionally, the expression level of RFC4 predicted radiotherapy responsiveness and the outcome of neoadjuvant radiotherapy in LARC patients.