Genome-wide RNAi Screening Identifies RFC4 as a Factor That Mediates Radioresistance in Colorectal Cancer by Facilitating Nonhomologous End Joining Repair.

Abstract

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