Clinical investigation
Breast
Intact Mre11/Rad50/Nbs1 Complex Predicts Good Response to Radiotherapy in Early Breast Cancer

https://doi.org/10.1016/j.ijrobp.2006.12.005Get rights and content

Purpose

To investigate the expression and predictive role of the Mre11/Rad50/Nbs1 (MRN) complex and the ataxia-telangiectasia mutated protein (ATM) for the outcome of radiotherapy in breast cancer patients.

Methods and Materials

The protein expression of ATM and the DNA repair proteins in the MRN complex were investigated using immunohistochemistry in tumors from 224 women with early breast cancer, who were randomized to receive postoperative radiotherapy or adjuvant chemotherapy.

Results

Compared with normal breast tissue, the staining intensity of Mre11, Rad50, Nbs1, and ATM was reduced in a majority of the tumors. Weak expression of the MRN complex was correlated with high histologic grade and estrogen receptor negativity (p = 0.01 and p = 0.0001, respectively). Radiotherapy significantly reduced the risk of local recurrence as compared with chemotherapy (p = 0.04). The greatest benefit of radiotherapy was seen in patients with moderate/strong expression of the MRN complex (relative risk = 0.27, 95% confidence interval = 0.098–0.72, p = 0.009), whereas patients with negative/weak MRN expression had no benefit of radiotherapy compared with adjuvant chemotherapy. These results suggest that an intact MRN complex is important for the tumor cell eradicating effect of radiotherapy.

Conclusions

Reduced expression of the MRN complex predicts a poor effect of radiotherapy in patients with early breast cancer.

Introduction

Adjuvant radiotherapy significantly reduces the risk of locoregional recurrence in breast cancer (1, 2). However, many patients have local relapses despite postoperative radiation treatment. An improved understanding of the mechanisms behind this tumor cell resistance to radiotherapy increases the possibility of offering an individualized treatment that would further reduce the risk of local recurrence.

Ionizing radiation used in radiotherapy induces different kinds of damage, but it is the double-strand breaks that best correlate with cell killing (3, 4, 5). Double-strand breaks of the DNA are repaired by three different mechanisms: single-strand annealing, homologous recombination, and nonhomologous end-joining, of which the latter two are considered the most important (4).

One of the central components in the signaling triggered by DNA double-strand breaks is the ataxia-telangiectasia mutated protein (ATM). Upon exposure to ionizing radiation, ATM is rapidly activated. The ATM protein phosphorylates a large number of critical targets that are involved in DNA repair, apoptosis, and cell cycle arrest (6). The ATM protein is mutated in patients with ataxia telangiectasia, a rare autosomal disease characterized by progressive neuronal degeneration, increased risk of cancer, and radiosensitivity (7).

Two other rare syndromes that closely resemble ataxia telangiectasia are ataxia telangiectasia–like disorder (ATLD) and Nijmegen breakage syndrome (NBS), both of which are associated with high cancer susceptibility and high sensitivity to ionizing radiation. Ataxia telangiectasia–like disorder and NBS are caused by germ-line mutations in the genes encoding Mre11 and Nbs1, respectively (8, 9). Mre11 and Nbs1 form a complex together with Rad50. The Mre11/Rad50/Nbs1 (MRN) complex is a vital component in the repair of double-strand breaks and is indicated in nonhomologous end-joining, due to interactions with ATM and Ku70. Nbs1 is involved in the intra-S-phase checkpoint, and the Nbs1 protein is one of the substrates phosphorylated by ATM (10). The temporal relationship between ATM and the MRN complex is, however, not yet fully understood, because the MRN complex may not only be a downstream effector of ATM but may also function in activating ATM. It was recently shown that the MRN complex acts as a double-strand break sensor and recruits ATM to broken DNA molecules (11). It has also been suggested that the function of the MRN complex in double-strand break repair is to link the broken DNA ends and bring them close together to enable DNA repair (12).

The aim of this study was to examine the protein expression patterns of ATM, Mre11, Rad50, and Nbs1 using immunohistochemistry. The prognostic and predictive role of these four DNA repair proteins was investigated in 224 breast cancer patients. Our hypothesis was that downregulated expression of these proteins in tumor cells leads to impaired repair of radiation-induced DNA damage in these cells. This could lead to better eradication of tumor cells and be reflected as improved local tumor control. This is, to our knowledge, the first study investigating the role of these DNA repair proteins in the outcome of radiotherapy in breast cancer patients.

Section snippets

Patients and tumors

The tumor material in this study comes from 224 premenopausal women included in a prospective clinical trial that compared postoperative radiotherapy with adjuvant chemotherapy (13). The patients had either histologically verified lymph node metastases or a tumor diameter exceeding 30 mm. Tumor and treatment characteristics of the patients are presented in Table 1. The tumors were removed by modified radical mastectomy, fixed in formalin, and embedded in paraffin. Postoperative radiotherapy was

Results

Normal breast epithelium showed strong nuclear staining for Mre11, Rad50, and Nbs1 and moderate staining intensity for ATM (Fig. 1). There was no cytoplasmic staining in the normal tissue for any of the proteins. The staining intensity for Mre11, Rad50, Nbs1, and ATM was reduced in a majority of the tumors (Fig. 1, Table 2). The percentage of positive nuclei was increased compared with normal breast tissue in 20% of the tumors for Mre11, 79% for Nbs1, 61% for Rad50, and 80% for ATM.

The nuclear

Expression of Mre11, Rad50, Nbs1, and ATM in breast cancer

Compared with normal breast tissue, the staining intensity of Mre11, Rad50, and Nbs1 was reduced in a large proportion of the tumors, which could indicate that downregulation or loss of these DNA repair proteins is a common event in breast cancer. Reduced expression of these proteins in breast tumors has also been shown in a previous study by Angéle et al. (15), but the underlying mechanisms are not yet understood. There was a good correlation among the expression of the three proteins of the

Acknowledgments

The authors thank Lilianne Ferraud and Birgitta Holmlund for constructing the tissue microarrays; Najme Wall and Jan Olofsson for classifying the tumors according to the Nottingham grading system; and Torsten Hägerström for collecting the archived tumor material.

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    Supported by grants from the Swedish Cancer Society (O.S., B.N., and M.S.A.).

    Conflict of interest: none.

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