A Comprehensive Analysis of Alterations in DNA Damage Repair Pathways Reveals a Potential Way to Enhance the Radio-Sensitivity of Esophageal Squamous Cell Cancer

Abstract

Esophageal squamous cell cancer (ESCC) is a common malignancy with a poor 5-year overall survival in China. Altered DNA damage repair (DDR) pathways are associated with a predisposition to cancer and contribute to therapeutic response and resistance in cancers. However, alterations of DDR pathway genes in ESCC are still largely unknown. In this study, we employed genome sequencing data of 192 samples, comparative genomic hybridization data of 123 cases, and gene expression microarray data of 119 patients to firstly perform a comprehensive analysis of the gene alterations of 7 DDR pathways in ESCC. Gene mutations and copy number variations (CNVs) were observed in all 7 DDR pathways, and especially, CNVs were the dominant alteration types. Compared with other pathways, two DNA double-strand break (DSB) repair pathways homologous recombination (HR) and non-homologous end joining (NHEJ), carried significant gene mutations and CNVs especially gene amplifications. Most genes including RAD54B, NBS1, RAD51B, and PRKDC were significantly amplified and over-expressed in ESCC. Amplification and high expression of DSB repair pathway genes were associated with poorer overall survival. Gene set variation analysis further showed that DSB repair pathways were up-regulated in ESCC. Besides, we firstly demonstrated that combination of mirin and NU7441, two inhibitors for HR and NHEJ respectively, with ionizing radiation treatment significantly enhanced DSBs, reduced clonogenic cell survival, inhibited cell proliferation, and promoted cell apoptosis in ESCC cells with DSB pathway gene amplification. These findings suggest that DSB repair pathways were significantly altered in ESCC and inhibiting DSB repair pathways might enhance the radio-sensitivity of ESCC with DSB repair up-regulation.

Keywords: DNA damage repair pathways; NU7441; esophageal squamous cell cancer; homologous recombination; mirin; non-homologous end joining; radio-sensitivity.

Figure 1

The workflow of data analysis in this study.

Figure 2

DNA damage repair (DDR) pathway genes were mutated in esophageal squamous cell cancer (ESCC). (A) A complex heatmap that shows the non-silent gene mutation profile in DDR pathways (genes and samples with no gene mutations are removed). The top panel presents the number of gene mutations in each of ESCC samples, and the right panel shows the number of gene mutations in each gene. (B) A bubble plot that depicts the gene mutation enrichment analysis result of DDR pathways.

Figure 3

DNA damage repair (DDR) pathway genes had significant copy number variations (CNVs) in esophageal squamous cell cancer (ESCC). (A) A complex heatmap that shows the gene CNV profile in DDR pathways (samples with no CNVs are removed). The top panel presents the number of gene CNVs in each of ESCC samples, and the right panel shows the number of gene CNVs in each gene. (B) Amplification of homologous recombination (HR), non-homologous end joining (NHEJ), and the “DSB repair pathway” was associated with poorer overall survival. The amplification of MRE11-RAD50-NBS1 (MRN) complex genes (C), RAD54B, and RAD51B (D) was related to shorter overall survival.

Figure 4

The messenger RNA (mRNA) expression level of double-strand break (DSB) repair pathway genes was up-regulated in esophageal squamous cell cancer (ESCC). (A) A heatmap that depicts the mRNA expression profile of DSB repair pathway genes in ESCC and normal tissues from GSE53624 dataset. (B) Student’s t-test analysis showed that RAD54B, RAD51B, MRE11, NBS1, and PRKDC were up-regulated in ESCC with statistically significant P values. ESCC patients were divided into two groups based on the median expression values of DSB repair pathway genes, and survival analysis was then performed. High expression of RAD51B, MUS81, TOP3A, GEN1, and TP53BP1 was associated with poorer overall survival of ESCC patients (C).

Figure 5

The activities of double-strand break (DSB) repair pathways were up-regulated in esophageal squamous cell cancer (ESCC) as determined by gene set variation analysis (GSVA). (A) A heatmap that shows the GSVA scores of homologous recombination (HR), non-homologous end joining (NHEJ), and the “DSB repair pathway” in each ESCC or normal sample. (B) Welch’s unequal variances t-test result showed that the activities of HR, NHEJ and the “DSB repair pathway” were significantly up-regulated in ESCC based on the GSVA scores. ESCC patients were divided into two groups based on the median GSVA scores of HR, NHEJ and the “DSB repair pathway” respectively, and survival analysis was then performed. High activities of HR, NHEJ, and the “DSB repair pathway” were associated with shorter overall survival (C). (D) Compared to ESCC patients with N = 0, the GSVA scores of NHEJ and the “DSB repair pathway” were significantly higher in ESCC cases with N > 0. Besides, the GSVA scores of NHEJ in ESCC samples of stage III were significantly higher than ESCC patients of stage I and II.

Figure 6

Combination of mirin and NU7441 with ionizing radiation (IR) treatment significantly enhanced double-strand breaks (DSBs) in esophageal squamous cell cancer (ESCC) cells. (A, B) DSBs were indicated by immunostaining with γ-H2AX. Combinations of mirin (50 µM) or/and NU7441 (5 µM) with IR (6 Gy) treatment significantly improved the number of γ-H2AX foci in both YES2 and KYSE30 cells. Scale bar = 30 µm. All the experiments were independently performed in triplicate. The error bars represent the standard deviation and P values were evaluated by Student’s t-test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s. P > 0.05.

Figure 7

Combination of mirin and NU7441 with ionizing radiation (IR) treatment significantly reduced clonogenic cell survival, inhibited cell proliferation and promoted cell apoptosis in esophageal squamous cell cancer (ESCC) cells. (A, B) Clonogenic cell survival with combinations of inhibitors and IR (6 Gy) treatment was investigated by clonogenic assay. Combinations of mirin (50 µM) or/and NU7441 (5 µM) with IR treatment significantly reduced number of colonies of both YES2 and KYSE30 cells. (C) Cell proliferation was measured at 24, 48, 72, and 96 h after treatment with inhibitors and IR (6 Gy) by MTS assay. Combinations of mirin (50 µM) or/and NU7441 (5 µM) with IR treatment significantly inhibited proliferation of both YES2 and KYSE30 cells. (D, E) Flow cytometric analysis was applied to detect the effect of combinations of inhibitors and IR (6 Gy) treatment on cell apoptosis. Combinations of mirin (50 µM) or/and NU7441 (5 µM) with IR treatment significantly promoted cell apoptosis of both YES2 and KYSE30 cells. All the experiments were independently performed in triplicate. The error bars represent the standard deviation and P values were evaluated by Student’s t-test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, n.s. P > 0.05.