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Supplementary Figure S1 shows the procedure to generate radioresistant HCT1166GyR cells; Supplementary Figure S2 shows reporter constructs of NHEJ and HR pathway; Supplementary Figure S3 shows representative images of IHC assays; Supplementary Figure S4 show the detailed data of screening candidate radioresistant genes; Supplementary Figure S5 shows RFC4 expression in CRC cells and tissues after radiation; Supplementary Figure S6 shows the representative clonogenic pictures for Figure 1C; Supplementary Figure S7 shows representative charts of Annexin V/PI double staining assays for Figure 1F; Supplementary Figure S8 shows the effects of RFC4 on γ-H2AX and pATM levels after radiation; Supplementary Figure S9 shows the additional evidences of RFC4 to promote NHEJ- but not HR-mediate DNA repair; Supplementary Figure S10 shows the effects of RFC4 on the chemotherapeutic drug resistance in HCT116 cells.

Supplementary Table S4 shows the correlation of clinicopathological parameters and RFC4 expression.

Supplementary Tables S5 & S6 show the candidate genes picked out in HCT116 and HT29 cells; Supplementary Tables S7 & S8 show the proteins might bind with RFC4, detected by IP-MS in HCT116 and HT29 cells.

Supplementary Table S1 shows the sequences of siRNAs; Supplementary Table S2 shows the informations of shRNAs; Supplementary Table S3 shows the sequences of quantitative PCR Primers.

Purpose: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.Experimental Design:A genome-wide RNAi screen was used to search for candidate radioresistance genes. After RFC4 knockdown or overexpression, colorectal cancer 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 patients with LARC receiving neoCRT.Results:RFC4, NCAPH, SYNE3, LDLRAD2, NHP2, and FICD were identified as potential candidate radioresistance genes. RFC4 protected colorectal cancer cells from X-ray–induced DNA damage and apoptosis in vitro and in vivo. Mechanistically, RFC4 promoted nonhomologous 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 patients with LARC 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 colorectal cancer cells. In addition, the expression level of RFC4 predicted radiotherapy responsiveness and the outcome of neoadjuvant radiotherapy in patients with LARC.

Purpose: 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. Experimental Design: A genome-wide RNAi screen was used to search for candidate radioresistance genes. After RFC4 knockdown or overexpression, colorectal cancer 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 patients with LARC receiving neoCRT. Results: RFC4, NCAPH, SYNE3, LDLRAD2, NHP2, and FICD were identified as potential candidate radioresistance genes. RFC4 protected colorectal cancer cells from X-ray–induced DNA damage and apoptosis in vitro and in vivo. Mechanistically, RFC4 promoted nonhomologous 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 patients with LARC 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 colorectal cancer cells. In addition, the expression level of RFC4 predicted radiotherapy responsiveness and the outcome of neoadjuvant radiotherapy in patients with LARC.

Chemoradiotherapy combined with surgical resection is the standard treatment for locally advanced rectal cancer, but not all the patients respond to neoadjuvant treatment. Transforming acidic coiled-coil protein-3 (TACC3) is frequently aberrantly expressed in rectal cancer tissue. In this study, we investigated whether TACC3 could serve as a biomarker predictive of the efficacy of chemoradiotherapy. In all, 152 rectal cancer patients with tumor tissue collected at biopsy and set aside before treatment were enrolled in this study. All patients received chemoradiotherapy and surgical resection. Immunohistochemically detected tumoral TACC3 expression significantly decreased sensitivity to chemoradiotherapy [risk ratio (RR) = 2.236, 95% confidence interval (CI): 1.447–3.456; P = 0.001] and thus the pathological complete response rate (P = 0.001). TACC3 knockdown using specific siRNA enhanced radiotherapy-induced decreases in proliferation and colony formation by HCT116 and SW480 cells and increased the incidence of radiotherapy-induced apoptosis. Cox multivariate analysis showed that TACC3 was a significant prognostic factor for overall survival (P = 0.017) and disease-free survival (P = 0.020). These findings suggest TACC3 expression may be predictive of chemoradiotherapy sensitivity and prognosis in locally advanced rectal cancer.

Carboxylesterase (CarE) is a multifunctional superfamily, and it plays important roles in xenobiotic detoxification, pheromone degradation, neurogenesis and regulating development. In this research, firstly, we measured the rutin-induced transcriptional level of BmCarE-10 gene by using real-time quantitative RT-PCR method, and dual spike-in strategy. Several possible physiological functions were certified preliminarily by RNAi experiments in silkworm. Promoter truncation analysis using a dual-luciferase reporter assay in Bombyx mori ovary cells (BmN) showed that the region -705 to -625 for BmCarE-10 gene was essential for basal and rutin-induced transcriptional activity. Sequence analysis of this region revealed several potential transcriptional regulatory elements such as Croc and Dfd. The activities of the BmCarE-10 promoter in various tissues of silkworm were also measured by firefly luciferase activity and normalized by the Renilla luciferase activity. Results showed that the activity of the BmCarE-10 promoter were highest in the Malpighian tubule, followed by fat body, silk gland, midgut, epidermis, and hemocyte. The essential region for basal and rutin-induced transcriptional activity was also -894 to -502 in Malpighian tubule and fat body of silkworm. The potential core promoters of BmCarE-10 gene in B. mori are reported for the first time in this research. Further identification of cis- and trans-elements and their role in upregulation of BmCarE-10 gene may be useful for elucidating the contribution of CarE protein to the response mechanism to rutin.