XRCC2 as a predictive biomarker for radioresistance in locally advanced rectal cancer patients undergoing preoperative radiotherapy

Abstract

XRCC2 has been shown to increase the radioresistance of some cancers. Here, XRCC2 expression was investigated as a predictor of preoperative radiotherapy (PRT) treatment response in locally advanced rectal cancer (LARC). XRCC2 was found to be overexpressed in rectal cancer tissues resected from patients who underwent surgery without PRT. In addition, overall survival for LARC patients was improved in XRCC2-negative patients compared with XRCC2-positive patients after treatment with PRT (P < 0.001). XRCC2 expression was also associated with an increase in LARC radioresistance. Conversely, XRCC2-deficient cancer cells were more sensitive to irradiation in vitro, and a higher proportion of these cells underwent cell death induced by G2/M phase arrest and apoptosis. When XRCC2 was knocked down, the repair of DNA double-strand breaks caused by irradiation was impaired. Therefore, XRCC2 may increases LARC radioresistance by repairing DNA double-strand breaks and preventing cancer cell apoptosis. Moreover, the present data suggest that XRCC2 is a useful predictive biomarker of PRT treatment response in LARC patients. Thus, inhibition of XRCC2 expression or activity represents a potential therapeutic strategy for improving PRT response in LARC patients.

Keywords: Clinical Section; XRCC2; preoperative radiotherapy; radioresistance; rectal cancer.

Figure 1. XRCC2 expression in the resected specimens that did not receive PRT

A. Expression levels of XRCC2 mRNA were higher in the 50 rectal cancer samples compared with the 50 corresponding normal colorectal mucosa tissue samples (Wilcoxon signed rank test, **P < 0.01). B. Expression of XRCC2 was detected by Western blotting and higher levels were present in the rectal cancer samples (T1-T4) compared with the matched adjacent non-tumor tissues (N1-N4). C., D. Representative images of rectal cancer tissues that were positive C. and negative D. for XRCC2 expression in the immunohistochemical analysis performed (×200 magnification). E. A representative image of normal colorectal mucosa tissue that was negative for XRCC2 expression by immunohistochemistry (×200 magnification).

Figure 2. Decreased XRCC2 expression in pretreatment biopsy tissue samples of LARC patients who received PRT is associated with improved postoperative histological tumor regression and better long-term prognosis

A. Positive XRCC2 expression in a representative biopsy specimens from a patient who exhibited resistance to PRT. B. Negative XRCC2 expression in a representative biopsy specimen from a patient who exhibited sensitivity to PRT. C. Positive XRCC2 expression in a representative biopsy specimen with poor tumor regression grade (TRG ≤ 2). D. Negative XRCC2 expression in a representative biopsy specimen with good tumor regression grade (TRG ≥ 3). E. Kaplan-Meier plot of overall survival. Survival was significantly improved in patients with XRCC2-negative tumors (blue line) than in patients with XRCC2-positive tumors (green line) (P < 0.01).

Figure 3. Successful knockdown of XRCC2 in SW480 colorectal cancer cells

A. Western blotting was used to detect XRCC2 knockdown efficiency in untransfected SW480 cells, SW480 cells transfected with nonsilencing (vector) shRNAs, and SW480 cells transfected with shRNA1 and shRNA2. B. Lower expression of XRCC2 mRNA was detected in XRCC2 knockdown SW480 cells using quantitative real-time PCR.

Figure 4. XRCC2 knockdown cells exhibit impaired repair of radiation-induced DNA DSBs

A. The levels of immunofluorescence that were observed in a phosphorylation of H2AX (γ-H2AX) assay. Images of γ-H2AX foci were obtained 0, 0.5, 6, and 24 h after 2 Gy of IR was applied to vector control cells and SW480 cells transduced with shRNA1. B. The average number of γ-H2AX foci per nucleus was calculated based on the number of γ-H2AX foci that were detected in more than 100 cells for each treatment group. The error bars represent the SD from three independent experiments. Statistical differences between the numbers of γ-H2AX foci in the control cells (SW480 and vector alone) and the shXRCC2 cells 6 h and 24 h after an IR treatment were calculated (*P < 0.05, **P < 0.01). C. Representative images of the clonogenic cell survival assays that were performed. IR treatment inhibited the colony-forming capacity of the cancer cells in a dose-dependent manner. Furthermore, sh-XRCC2 enhanced the tumor suppressive effect of the IR. D. The survival fraction curves for the SW480, vector, and sh-XRCC2 cells that were tested in clonogenic survival assays. Data shown are the mean ± SD of three independent experiments.

Figure 5. XRCC2 knockdown significantly increases cell apoptosis that is induced by IR treatment

A. Levels of apoptosis in the XRCC2 knockdown cells versus the control cells (SW480 and vector alone) that were treated with 6 Gy of IR for 24 h. The cells were stained with Annexin V and PI. B. The percentage of cells that underwent apoptosis following IR treatment were quantitated and the data are expressed as the mean ± SD from three independent experiments (*P < 0.05, **P < 0.01). C. Detection of PARP, caspase-9, cleaved caspase-3, and Bcl-2 protein levels by Western blotting. Arrow heads and stars represent the pro-forms and the cleaved forms of PARP and caspase 9, respectively. Detection of GAPDH was used as a loading control.

Figure 6. XRCC2 knockdown increases cell cycle arrest in the G2 phase and activates p-Chk2 after IR treatment

A. Flow cytometry analysis of the proportion of the cells indicated in each stage of the cell cycle (G1, G2, and S) after the IR treatment. B. The percentage of cells in the G2/M phase of the cell cycle were quantitated and expressed as the mean ± SEM from three independent experiments (*P < 0.01). C. Expression levels of the γ-H2AX, phosphorylated Chk2-Thr68, and Chk2 proteins that were detected by Western blotting in the SW480, vector, and Sh-XRCC2 cell lines. Expression levels of phosphorylated Chk2-Thr68 and -H2AX were higher in the sh-XRCC2 cells compared to the controls after IR. Detection of GAPDH was used as a loading control.