2024 Breast Cancer Risk Is Associated with the Genes Encoding the DNA Double-Strand Break Repair Mre11/Rad50/Nbs1 Complex Huan-Ming Hsu,1,2,3 Hui-Chun Wang,2 Sou-Tong Chen,6 Giu-Cheng Hsu,4 Chen-Yang Shen,2,5 and Jyh-Cherng Yu3 1Graduate Institute of Medical Sciences, National Defense Medical Center; 2Institute of Biomedical Sciences, Academia Sinica; Departments of 3Surgery and 4Radiology, Tri-Service General Hospital; 5Life Science Library, Academia Sinica, Taipei, Taiwan; and 6Department of Surgery, Changhua Christian Hospital, Changhua, Taiwan Abstract The evolutionarily conserved Mre11-Rad50-Nbs1 observations that (a) one single-nucleotide polymor- (MRN) complex, consisting of proteins encoded by phism in Nbs1 was significantly associated with breast the genes Mre11, Rad50, and Nbs1, was recently shown cancer risk, and a trend toward an increased risk of to play a crucial role in DNA double-strand break developing breastcancer was found in women harbor- (DSB) repair by recruiting the nuclear protein kinase ing a greater number of putative high-risk genotypes of ataxia telangiectasia mutated to DSB sites, leading to MRN genes (an adjusted odds ratio of 1.25 for each activation of this DNA repair network. Given the fact additional putative high-risk genotype; 95% confidence that carriers of defective mutation and polymorphic interval, 1.10-1.44); (b) this association between risk and variants of ataxia telangiectasia mutated are athigher the number of putative high-risk genotypes was risk of developing breastcancer, we hypothesizeda stronger and more significant in women thought to be role of the MRN genes in determining breast cancer more susceptible to estrogen, i.e., those with no history susceptibility. This hypothesis was examined both in a of full-term pregnancy, those older (z26 years of age) at case control study of 559 breast cancer patients and first full-term pregnancy, or those having had fewer 1,125 healthy women of single-nucleotide polymor- (<2) full-term pregnancies; the risk effect conferred by phisms in Mre11, Rad50, and Nbs1 and by the in vivo harboring a higher number of high-risk genotypes of detection of binding between Mre11 and BRCA1, MRN genes was more significantin women withouta encoded by the breast cancer susceptibility gene history of breast feeding; and (c) Mre11 and BRCA1 BRCA1. We were also interested in defining whether were shown to form a complex in vivo, and this any association between MRN genes and breast cancer interaction was increased by irradiation. This study was modified by reproductive risk factors reflecting the supports the role of the MRN pathway in breast cancer level of estrogen exposure or susceptibility to estrogen development, further strengthening the suggestion that exposure, as estrogen is known to initiate breast cancer mechanisms regulating DSB repair may play a mutator development due to its metabolites causing DSB role driving breastcancer pathogenesis. (Cancer formation. Support for the hypothesis came from the Epidemiol Biomarkers Prev 2007;16(10):2024–32) Introduction Double-strand breaks (DSB) are extremely cytotoxic specific partner proteins. Recently, the evolutionarily DNA lesions, and cells have therefore developed an conserved Mre11/Rad50/Nbs1 (MRN) complex was extensive array of responses that lead to damage repair, implicated in ATM recruitment to DSBs (6-8). This thus preventing cell death (1-4). The nuclear protein complex is involved in the initial processing of DSBs kinase ataxia telangiectasia mutated (ATM) is regarded due to its nuclease activity and DNA binding capability, as the primary activator of this network, and the which reside in the Mre11 protein and partially depend recruitment of ATM to DNA DSBs is thought to be the on the interaction of Mre11 with Rad50, which provides critical step in its activation and function (5). However, the energy source for the MRN complex (9, 10). However, although ATM has an affinity for DNA, this recruitment in terms of the protein required for ATM recruitment, has been suggested to require and to be facilitated by it has only recently been recognized that Nbs1 recruits activated ATM to sites of DNA damage, then promotes its phosphorylation and the triggering of subsequent Received 2/7/07; revised 7/18/07; accepted 7/27/07. steps in the DNA damage response (11). These results The costs of publication of this article were defrayed in part by the payment of page show that the MRN complex functions as a DNA DSB charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734solely to indicate this fact. sensor upstream of ATM signaling. However, this critical Requests for reprints: Chen-Yang Shen, Institute of Biomedical Sciences, Academia role of the complex in initiating DSBs repair does not Sinica, Taipei, 11529, Taiwan. Fax: 886-2-2782-3047. E-mail: [email protected] exclude its involvement in mechanisms downstream or Jyh-Cherng Yu, Departments of Surgery, Tri-Service General Hospital, Taipei, 11529, Taiwan. Phone: 886-2-8792-7191. E-mail: [email protected] of ATM signaling in the intra-S or G2-M cell cycle Copyright D 2007 American Association for Cancer Research. checkpoint in response to DNA damage, as suggested in doi:10.1158/1055-9965.EPI-07-0116 earlier studies (9, 10). These functional links between Cancer Epidemiol Biomarkers Prev 2007;16(10). October 2007 Downloaded from cebp.aacrjournals.org on September 26, 2021. © 2007 American Association for Cancer Research. Cancer Epidemiology, Biomarkers & Prevention 2025 ATM and the MRN complex help explain similarities in ing the effects of subtle genetic variations, such as single the clinical and cellular phenotypes associated with nucleotide polymorphisms (SNP). Furthermore, the deficiency of each of the genes encoding these proteins. use of a genetically homogenous population (i.e., the Hypomorphic mutations in Mre11 and Nbs1 result, Taiwanese population) reduces the chance of false respectively, in the human genetic instability disease, positives due to population stratification (25, 26). The ataxia-telangiectasia–like disorder, and Nijmegen break- present study included 559 female breast cancer patients age syndrome (10, 12, 13). These single-gene disorders and 1,125 healthy female controls. All subjects gave their overlap with one another and with ataxia-telangiectasia, informed consent. The recruitment of both cases and caused by ATM deficiency, all being characterized by controls and considerations regarding methodologic neurologic abnormalities, radiosensitivity, impairment of issues (such as study design, sampling scheme, and the cellular response to DSBs, and genomic instability. potential bias) have been described and addressed in The close relationship between ATM and the MRN detail previously (16, 19, 20, 25, 26). complex prompted us to hypothesize a role of the MRN Questionnaire. Two experienced research nurses were genes in determining breast cancer susceptibility. The assigned to administer a structured questionnaire to both rationale underlying this hypothesis is that carriers cases and controls. The information collected and the of either defective mutations or polymorphic variants validity of this questionnaire have been addressed and of ATM are at higher risk of developing breast cancer confirmed in our previous studies (16, 19, 20, 25, 26). (14, 15). Furthermore, we have adopted a model (i.e., the hide-then-hit hypothesis; ref. 16) to explain the lack of Genotyping. Genomic DNA was extracted from the cancer predisposition in ataxia-telangiectasia–like disor- buffy coat isolated from whole-blood samples using a der patients or no breast cancer phenotype observed in QIAamp DNA extraction kit (Qiagen, Inc.) following the manufacturer’s protocol. Nijmegen breakage syndrome patients and suggest that a 7 disparate spectrum of disease phenotypes can be Using the current data from HapMap on haplotype differently caused by mutated forms or hypomorphic/ blocks of MRN genes in the Chinese population, we polymorphic variants of the same genes. This is because, selected SNPs in each block to detect genetic variation in in contrast to common genetic diseases, cancer formation these three candidate genes and five SNPs were selected requires an extended period of time for the essential for each MRN gene. These SNPs were chosen because genomic changes to accumulate. Genetic variants or low- they are evenly distributed throughout the entire genes, penetrance alleles of DNA repair genes (such as Mre11/ a total of 15 SNPs being genotyped (Mre11: rs535801, RAD50/NBS1 examined in the present study) may rs569143, rs601391, rs684507, and rs1061945; Nbs1: therefore have a chance to escape the lethality phenotype rs1805794, rs1805790, rs709816, rs1061302, and by not triggering obvious cell cycle surveillance, and the rs1063045; Rad50: rs2252775, rs2301713, rs3798134, cells accumulate the necessary genomic instability rs2240032, and rs2244012). Because there have not been leading to cancer development. In addition, the possibil- any reports of an association between genotypic and ity of manifesting the tumorigenic phenotype depends phenotypic changes in the SNPs of the MRN genes, these not only on the joint effect of individual genes but also on selected SNPs were used as markers to reflect possible the interaction between genes and risk factors. To test linkage disequilibrium (LD) between themselves and this hypothesis, we did this investigation based (a)on different alleles of a gene of unidentified phenotypic a case control study to estimate the breast cancer risk variation. We used more than one SNP per gene to have associated with harboring putative high-risk genotypes an unbiased definition of the allelic and haplotypic of MRN genes, (b) on an examination of the joint effect statuses of each gene. of MRN genotypes and well-established risk factors of All SNPs were genotyped using a MassARRAY breast cancer in determining cancer risk, and (c)onan (SEQUENOM, Inc.). The PCR primers and extension in vivo study of the interaction between MRN and BRCA1, primers for all SNPs were designed using Spectro- encoded by the breast cancer susceptibility gene BRCA1.