Enhanced RAD21 cohesin expression confers poor prognosis and resistance to chemotherapy in high grade luminal, basal and HER2 breast cancers

Abstract

Introduction: RAD21 is a component of the cohesin complex, which is essential for chromosome segregation and error-free DNA repair. We assessed its prognostic and predictive power in a cohort of in situ and invasive breast cancers, and its effect on chemosensitivity in vitro.

Methods: RAD21 immunohistochemistry was performed on 345 invasive and 60 pure in situ carcinomas. Integrated genomic and transcriptomic analyses were performed on a further 48 grade 3 invasive cancers. Chemosensitivity was assessed in breast cancer cell lines with an engineered spectrum of RAD21 expression.

Results: RAD21 expression correlated with early relapse in all patients (hazard ratio (HR) 1.74, 95% confidence interval (CI) 1.06 to 2.86, P = 0.029). This was due to the effect of grade 3 tumors (but not grade 1 or 2) in which RAD21 expression correlated with early relapse in luminal (P = 0.040), basal (P = 0.018) and HER2 (P = 0.039) groups. In patients treated with chemotherapy, RAD21 expression was associated with shorter overall survival (P = 0.020). RAD21 mRNA expression correlated with DNA copy number, with amplification present in 32% (7/22) of luminal, 31% (4/13) of basal and 22% (2/9) of HER2 grade 3 cancers. Variations in RAD21 mRNA expression in the clinical samples were reflected in the gene expression data from 36 breast cancer cell lines. Knockdown of RAD21 in the MDA-MB-231 breast cancer cell line significantly enhanced sensitivity to cyclophosphamide, 5-fluorouracil and etoposide. The findings for the former two drugs recapitulated the clinical findings.

Conclusions: RAD21 expression confers poor prognosis and resistance to chemotherapy in high grade luminal, basal and HER2 breast cancers. RAD21 may be a novel therapeutic target.

Figure 1

RAD21 immunohistochemistry in DCIS and invasive carcinoma. A, Strong nuclear RAD21 staining in DCIS. Scale bar = 20 μm. B, Strong nuclear RAD21 staining in an invasive carcinoma, luminal type. Scale bar = 20 μm. C, Absence of nuclear staining in an invasive carcinoma, basal type. Scale bar = 20 μm.

Figure 2

Kaplan-Meier curves stratified by nuclear RAD21 expression, relapse-free survival (A-F) and overall survival (G-H). Relapse free survival: A, in all tumors, (P = 0.009) (n = 247); B, grade 3 cancers (P = 0.023) (n = 117); C, grade 1 and 2 cancers (P = 0.342) (n = 130); D, grade 3 luminal cancers (P = 0.040) (n = 32); E, grade 3 basal cancers (P = 0.018) (n = 29); F, grade 3 HER2 cancers (n = 34); Overall survival: G, without chemotherapy (P = 0.779) (n = 148). H, treated with chemotherapy (P = 0.020) (n = 91).

Figure 3

Expression of RAD21 in breast cancer cell lines. A. Quantitative real time RT-PCR of RAD21 transcripts in human breast cancer cell lines. The expression level in MCF10A was used as a reference and given an arbitary value of 1. Relative expression of RAD21 gene was compared with MCF10A. Error bar = standard error mean (SEM). B. OmniViz Treescape showing the hierarchical clustering of the top 25 genes that correlated best with the two RAD21 gene probes (200607_s_at and 200608_s_at). Gene expression levels: red, up-regulation compared with the geometric mean; green, down-regulation compared with the geometric mean. The color intensity correlates with the degree of change. Raw dataset (GEO:GSE16795) was sourced from GEO [20].

Figure 4

Validation of RAD21 expression in MDA-MB-231 cells carrying shRAD21 knockdown constructs. A. Quantitative real time PCR analysis of RAD21 expression in clones stably expressing two different shRAD21 constructs and shRNAmir vector, relative to the parental cell line. B. Western blot analysis of RAD21 protein level. RAD21 expression in five independently derived clonal cell lines with two different stable RAD21 knockdown constructs was compared to the parental cell line, cells transfected with shRNAmir vector and an immortalized human breast epithelial cell line MCF10A. Pan-actin was used as loading control. A reduction in the levels of RAD21 protein in three stable clones (sh223_sc1, sh224_sc4 and sh224_sc5) was verified by semi-quantitative Western blot analysis (bottom panel). The levels of RAD21 protein were normalized to the pan-actin loading control and expressed as the percentage of the parental line where the RAD21 expression was given an arbitrary value of 100%. The values represent the mean of four independent experiments except for shRNAmir vector and MCF10A where three independent experiments were performed. Clones with a significant reduction in either RAD21 mRNA or protein levels were marked by asterisks (* P < 0.05; ** P < 0.005, Student t-test). Error bar = SEM.

Figure 5

Effect of shRNA-mediated RAD21 knockdown on cellular sensitivity to anti-cancer drugs. Independent cell clones with stably reduced RAD21 expression were derived from the breast cancer cell line, MDA-MB-231. Relative levels of RAD21 gene expression when compared to the parental line by qRT-PCR and semi-quantitative Western blot analysis were shown in Figure 4. Error bar = SEM. Clonogenic survival following treatment with: A. cyclophosphamide: sh223_sc1 (P = 0.0316), sh223_sc3 (P = 0.175), sh224_sc4 (P = 0.0187) and sh224_sc5 (P = 0.0563); B. 5-FU: sh223_sc1 (P = 0.020), sh224_sc4 (P = 0.0257) and sh224_sc5 (P = 0.0242); and C. etoposide: sh223_sc1 (P = 0.020) and sh224_sc5 (P = 0.042).