High RAD51 gene expression is associated with aggressive biology and with poor survival in breast cancer

Abstract

Purpose: Although the DNA repair mechanism is important in preventing carcinogenesis, its activation in established cancer cells may support their proliferation and aggravate cancer progression. RAD51 cooperates with BRCA2 and is essential in the homologous recombination of DNA repair. To this end, we hypothesized that RAD51 gene expression is associated with cancer cell proliferation and poor prognosis of breast cancer (BC) patients.

Methods: A total of 8515 primary BC patients with transcriptome and clinical data from 17 independent cohorts were analyzed. The median value was used to divide each cohort into high and low RAD51 expression groups.

Results: High RAD51 expression enriched the DNA repair gene set and was correlated with DNA repair-related genes. Nottingham histological grade, Ki67 expression and cell proliferation-related gene sets (E2F Targets, G2M Checkpoint and Myc Targets) were all significantly associated with the high RAD51 BC group. RAD51 expression was positively correlated with Homologous Recombination Deficiency, as well as both mutational burden and neoantigens that accompanied a higher infiltration of immune cells. Primary BC with lymph node metastases was associated with high expression of RAD51 in two cohorts. There was no strong correlation between RAD51 expression and drug sensitivity in cell lines, and RAD51 expression was lower after the neoadjuvant chemotherapy compared to before the treatment. High RAD51 BC was associated with poor prognosis consistently in three independent cohorts.

Conclusion: RAD51 gene expression is associated with aggressive cancer biology, cancer cell proliferation, and poor survival in breast cancer.

Keywords: BRCA; Breast cancer; DNA repair; HRD; Neoadjuvant chemotherapy; RAD51.

Figure 1.. Association between RAD51 gene expression and DNA repair.

(a) RAD51 expression between normal breast and tumor tissues in TCGA. n; normal breast, t; tumor tissues. (b) The scatter plots between RAD51 gene expression and Intratumoral heterogeneity (left) and Homologues Recombination Deficiency (HRD) score (right) in TCGA. (c) The enrichment plots of DNA repair pathway in gene enrichment analysis (GSEA) comparing high vs low expression of RAD51 divided by a median cut-off in TCGA, METABRIC, and GSE96058 cohorts. The boxplots show the expression of DNA repair-related genes; BRCA1, BRCA2, E2F1, E2F4, E2F7, and CDK12 by high vs low expression of RAD51. FDR less than 0.25 is regarded as significant in GSEA. * = p-value of statistical significance. The r-value indicates Spearman’s rank correlation coefficient. All two group comparisons are tested by Wilcoxon signed-rank test. The error bars in each boxplot show the 95% confidence interval. The line in the box shows the median, and top and bottom show the 25th and 75th percentiles respectively.

Figure 2.. Association between RAD51 and cancer cell proliferation.

(a) The scatter plot of RAD51 gene expression and proliferation score in TCGA. (b) The boxplots of RAD51 gene expression by Nottingham histological grade in TCGA, METABRIC, and GSE96058 cohorts. The scatter plots of MKI67 and RAD51 gene expressions. (c) GSEA of all cell proliferation related gene sets by the high and low expression of RAD51 with a median cut-off in TCGA, METABRIC, and GSE96058 cohort. FDR less than 0.25 is regarded as significant in GSEA. *= p-value of statistical significance. The r-value indicates Spearman’s rank correlation coefficient. All multiple group comparisons are tested by Kruskal–Wallis test. The error bars in each boxplot show the 95% confidence interval. The line in the box shows the median, and top and bottom show the 25th and 75th percentiles respectively.

Figure 3.. Association of RAD51 expression with mutation rates and BRCA mutations.

(a) The boxplots of silent and non-silent mutation rate by the high and low RAD51 expression with a median cut-off in TCGA. (b) The boxplots of RAD51 gene expression in BRCA1, BRCA2, and both wild-type and mutant breast cancer in TCGA and METABRIC. The number of patients with BRCA1, BRCA2 or either mutation was 30, 29, 57 in TCGA, and 55, 60, 114 in METABRIC. *= p-value of statistical significance. All two group comparisons are tested by Wilcoxon signed-rank test. The error bars in each boxplot show the 95% confidence interval. The line in the box shows the median, and top and bottom show the 25th and 75th percentiles respectively.

Figure 4.. RAD51 expression and immune activation and immune cell infiltration.

(a) The boxplots show immune activity scores from TCGA. (b) Immune cell infiltrations by the high and low RAD51 expression with a median cut-off in TCGA, METABRIC, and GSE96058 cohort. *= p-value of statistical significance. All two group comparisons are tested by Wilcoxon signed-rank test. The error bars in each boxplot show the 95% confidence interval. The line in the box shows the median, and top and bottom show the 25th and 75th percentiles respectively.

Figure 5.. Relationship between RAD51 and clinicopathological factors.

RAD51 gene expression by immunohistochemical subtype, stage, lymph node metastasis, and distant metastasis in TCGA, METABRIC, and GSE96058 cohorts. *= p-value of statistical significance. All two group comparisons are tested by Wilcoxon signed-rank test, and multiple groups by Kruskal–Wallis test. The error bars in each boxplot show the 95% confidence interval. The line in the box shows the median, and top and bottom show the 25th and 75th percentiles respectively.

Figure 6.. Relationship between RAD51 and drug response in breast cancer.

(a) The scatter plots of correlation between RAD51 expression and area under the curve (AUC) of each drug. Docetaxel and cisplatin are from the PRISM primary screen; PARP inhibitors are from the GDSC. (b) Boxplots show RAD51 expression before (light purple boxes) and after (dark purple boxes) NAC. (c) All boxplots compare RAD51 expression by immunohistochemical subtype between the two groups, orange for pCR: pathological complete response and light green for RD: residual tumor. *= p-value of statistical significance. The r-value indicates Spearman’s rank correlation coefficient. All two group comparisons are tested by Wilcoxon signed-rank test. The error bars in each boxplot show the 95% confidence interval. The line in the box shows the median, and top and bottom show the 25th and 75th percentiles respectively.

Figure 7.. Survival analyses by RAD51 expression.

Kaplan-Meier survival curves of the DFS, DSS and, OS by RAD51 high vs. low expressions with a median cut-off in TCGA, METABRIC, and OS of the GSE96058 cohort. High groups are indicated by red lines, low groups by blue lines. *= p-value of statistical significance. Log-rank test was used to test the significance of the survival analysis. The r-value indicates Spearman’s rank correlation coefficient.