Aberrations of the MRE11-RAD50-NBS1 DNA damage sensor complex in human breast cancer: MRE11 as a candidate familial cancer-predisposing gene

Abstract

The MRE11, RAD50, and NBS1 genes encode proteins of the MRE11-RAD50-NBS1 (MRN) complex critical for proper maintenance of genomic integrity and tumour suppression; however, the extent and impact of their cancer-predisposing defects, and potential clinical value remain to be determined. Here, we report that among a large series of approximately 1000 breast carcinomas, around 3%, 7% and 10% tumours showed aberrantly reduced protein expression for RAD50, MRE11 and NBS1, respectively. Such defects were more frequent among the ER/PR/ERBB2 triple-negative and higher-grade tumours, among familial (especially BRCA1/BRCA2-associated) rather than sporadic cases, and the NBS1 defects correlated with shorter patients' survival. The BRCA1-associated and ER/PR/ERBB2 triple-negative tumours also showed high incidence of constitutively active DNA damage signalling (gammaH2AX) and p53 aberrations. Sequencing the RAD50, MRE11 and NBS1 genes of 8 patients from non-BRCA1/2 breast cancer families whose tumours showed concomitant reduction/loss of all three MRN-complex proteins revealed two germline mutations in MRE11: a missense mutation R202G and a truncating mutation R633STOP (R633X). Gene transfer and protein analysis of cell culture models with mutant MRE11 implicated various destabilization patterns among the MRN complex proteins including NBS1, the abundance of which was restored by re-expression of wild-type MRE11. We propose that germline mutations qualify MRE11 as a novel candidate breast cancer susceptibility gene in a subset of non-BRCA1/2 families. Our data have implications for the concept of the DNA damage response as an intrinsic anti-cancer barrier, various components of which become inactivated during cancer progression and also represent the bulk of breast cancer susceptibility genes discovered to date.

Figure 1

Immunoperoxidase detection of MRN complex proteins and γH2AX in normal human breast and breast carcinomas. Normal human breast tissue shows strong nuclear positivity for all three proteins of the MRN complex: Mre11, Rad50 and Nbs1, while the staining for the DNA damage signalling marker γH2AX is negative. The two examples of invasive breast carcinomas shown in the middle and right‐hand columns also express the three MRN complex proteins, and the cancer cells are heterogeneously positive for the γH2AX marker, indicating ongoing DNA damage signalling.

Figure 2

Immunoperoxidase staining for MRN complex proteins and γH2AX in a ductal carcinoma in situ of the breast. Example of an in situ breast tumour with combined aberrant loss of all three MRN complex proteins, shown at low (left) and high (right) magnification. Note the preserved nuclear staining for the MRN proteins in the stromal cells and myoepithelial cells surrounding the tumour cells in the centre of the aberrant duct(s). This tumour shows very little if any staining for the γH2AX marker.

Figure 3

Immunoperoxidase detection of MRN complex proteins and γH2AX in an invasive ductal carcinoma of the breast. Example of an invasive breast tumour with aberrant selective loss of the Nbs1 protein, in contrast to largely preserved nuclear expression of Rad50 and Mre11 (variable), as shown at low (left) and high (right) magnification. Note the preserved nuclear staining for Nbs1 in the scattered stromal cells within the tumour, contrasting with virtually negative cancer cells. This tumour shows moderate yet detectable (heterogeneous) staining for the γH2AX marker, visible as foci in some nuclei at the higher magnification.

Figure 4

MRN defects are more frequent in BRCA1/2 and ER/PR/ERBB2‐triple‐negative breast tumours, and NBS1 deficiency associates with poor survival. (A) MRN protein expression is more frequently aberrant in BRCA1 and BRCA2 tumours than in nonBRCA1/2 tumours; with MRE11 defects in 30% (14/47) and 14% (6/42) vs. 7% (68/932), p=0.00001 and p=0.13, respectively; NBS1 defective in 26% (11/43) and 25% (10/40) vs. 11% (99/921), p=0.006 and p=0.01, respectively; and RAD50 defects in 32% (14/44) and 30% (12/40) vs. 3% (30/1059), p=0.000000001 and p=0.000000005, respectively. (B) MRN protein expression in ER/PR/ERBB2‐triple‐negative and other nonBRCA1/2 breast carcinomas. MRN defects are more common in the ER/PR/ERBB2‐triple‐negative subset of breast tumours than in other nonBRCA1/2 tumours; with MRE11 defects in 16% (19/119) vs. 6% (43/749), p=0.0003; NBS1 defects in 20% (24/122) vs. 9% (67/737), p=0.001; and RAD50 defects in 6% (8/140) vs. 2% (20/844), p=0.05. (C) Tumours defective in all three MRN complex proteins among BRCA1‐tumours: 23% (9/40), BRCA2‐tumours: 12% (4/33) and nonBRCA1/2‐tumours 3% (24/870), with p=0.000004 and p=0.016, respectively. (D) Breast cancer‐specific survival of patients with NBS1‐aberrant versus NBS1‐normal tumours; with cumulative survival at a 10‐years (120 months) follow‐up being 64% and 84%, respectively (p=0.0002).

Figure 5

Germline mutations of MRE11 in breast cancer families. (A) Schematic representation of the MRE11 protein with indicated functional domains and the position of the two germline heterozygous mutations identified among the 8 patients whose tumours showed concomitant reduction or loss of all three MRN complex proteins. The lower part documents the aberrantly lost MRN proteins in the tumour cells of the patient with the R633STOP mutation, in contrast to preserved MRN expression in stromal cells. (B) The 5‐generation pedigree of the breast cancer family of the patient (marked by arrow) with the germline R633STOP mutation. Note 3 cases of breast cancer and multiple other tumour types among the family members, all among maternal relatives.

Figure 6

Western blot analysis of MRN complex proteins in diverse human cell types and cancer cell lines. (A) Selective NBS1 protein defects in cells from NBS patients (NBS‐Tert fibroblasts and NBS‐1LBI lymphoblasts), reconstituted upon re‐expression of wild‐type NBS1: NBS‐Tert+Nbs1 and NBS‐1LBI+Nbs1. Expression of MRN protein complex in primary fibroblasts BJ and osteosarcoma cell line U‐2‐OS is normal, while the colon cancer cells HT29 show selective deficiency of NBS1. Cdk7 detection was used as a loading control. (B) All three MRN complex proteins are aberrant in the A‐TLD1 and A‐TLD2 fibroblasts, expressing the prematurely truncated Mre11 protein R633X, while the A‐TLD3 lymphoblasts (expressing Mre11 with the point missense mutation 350 A→G, Stewart et al., 1999) show only mild reduction of the Rad50 and Nbs1 protein abundance, compared to control normal human lymphoblasts (Nh‐LB). Cdk7 was used to verify equal loading. (C) MRN complex protein expression is normal in the MRN‐proficient colon cancer cell line SW620 (Giannini et al., 2002), while the HCT116 cells show low levels of endogenous full‐length Mre11 and a shorter dominant‐negative splice variant of Mre11 (Wen et al., 2008, and data not shown), with a strong impact on Nbs1 total abundance. Note that the level of endogenous Nbs1 is restored upon moderate expression of additional wild‐type Mre11 in HCT116 cells, and independently of the p53 status. Detection for SMC1 served as a loading control.

Figure 7

Immunofluorescence and confocal microscopy analysis of MRN complex proteins and γ‐H2AX in A‐TLD2 and U2OS cells. Panels of images documenting nuclear expression of the indicated proteins (red signal) in control U2OS cells, in contrast to the lack of MRN protein expression in A‐TLD2 cells with the homozygous Mre11 R633X mutation. Parallel DNA staining (DAPI, blue) and immunocytochemical staining for 53BP1 served to visualize nuclei and provide a positive protein control, respectively. The lower panels show a preserved ability to form the γ‐H2AX foci at 1h after 4Gy of ionizing radiation (IR) also in the A‐TLD2 cells.

Figure 8

Graph summary of constitutive gH2AX expression and p53 aberrations in various subsets of breast carcinomas. (A) Histone H2AX phosphorylation and p53 immunopositivity in BRCA1 and BRCA2 tumours vs. nonBRCA1/2 tumours, showing γH2AX positivity in 24/41 (59%) of BRCA1; 13/44 (30%) of BRCA2, and 468/978 (48%) in nonBRCA1/2 tumours, with the p values: p=0.2 for the BRCA1 vs. nonBRCA1/2, and p=0.0002 for BRCA2 vs. nonBRCA1/2. B) Constitutive DNA damage signalling (γH2AX) occurs more frequently in ER/PR/ERBB2‐triple‐negative breast cancers than in tumours with at least one of the hormone receptors or ERBB2 overexpression: 86/129 (67%) vs. 343/773 (44%), p=0.000003. The ER/PR/ERBB2‐triple‐negative tumours are also more frequently p53 aberrant than the other nonBRCA1/2 tumours (p<0.0000000001).

Figure 9

Schematic illustration of the roles of the known breast cancer susceptibility genes within the DNA damage response network. The 12 breast cancer susceptibility genes with known functions share the involvement of their protein products in the genome maintenance network, operating at various levels from sensing the DNA lesions (the MRN complex), through transducing the damage signal (ATM and Chk2), up to effector roles in DNA repair, checkpoint or cell death pathways (blue arrows). The previously reported breast cancer susceptibility genes are marked in yellow, the new candidate reported in this study (Mre11) in red, and the essential genes ATR and Chk1 that may contribute to tumorigenesis through haploinsufficiency in white. The PTEN tumour suppressor regulates DNA damage signalling and repair through regulation of Chk1 and Rad51 (dashed arrows), and Rad51 (shown in grey) might be a cancer susceptibility gene itself (see Section 3 for more details).