Chk2-dependent phosphorylation of XRCC1 in the DNA damage response promotes base excision repair.

The DNA damage response (DDR) has an essential function in maintaining genomic stability. Ataxia telangiectasia-mutated (ATM)-checkpoint kinase 2 (Chk2) and ATM- and Rad3-related (ATR)-Chk1, triggered, respectively, by DNA double-strand breaks and blocked replication forks, are two major DDRs processing structurally complicated DNA damage. In contrast, damage repaired by base excision repair (BER) is structurally simple, but whether, and how, the DDR is involved in repairing this damage is unclear. Here, we demonstrated that ATM-Chk2 was activated in the early response to oxidative and alkylation damage, known to be repaired by BER. Furthermore, Chk2 formed a complex with XRCC1, the BER scaffold protein, and phosphorylated XRCC1 in vivo and in vitro at Thr(284). A mutated XRCC1 lacking Thr(284) phosphorylation was linked to increased accumulation of unrepaired BER intermediate, reduced DNA repair capacity, and higher sensitivity to alkylation damage. In addition, a phosphorylation-mimic form of XRCC1 showed increased interaction with glycosylases, but not other BER proteins. Our results are consistent with the phosphorylation of XRCC1 by ATM-Chk2 facilitating recruitment of downstream BER proteins to the initial damage recognition/excision step to promote BER.

MicroRNA-30a inhibits cell migration and invasion by downregulating vimentin expression and is a potential prognostic marker in breast cancer.

Tumor recurrence and metastasis result in an unfavorable prognosis for cancer patients. Recent studies have suggested that specific microRNAs (miRNAs) may play important roles in the development of cancer cells. However, prognostic markers and the outcome prediction of the miRNA signature in breast cancer patients have not been comprehensively assessed. The aim of this study was to identify miRNA biomarkers relating to clinicopathological features and outcome of breast cancer. A miRNA microarray analysis was performed on breast tumors of different lymph node metastasis status and with different progression signatures, indicated by overexpression of cyclin D1 and ß-catenin genes, to identify miRNAs showing a significant difference in expression. The functional interaction between the candidate miRNA, miR-30a, and the target gene, Vim, which codes for vimentin, a protein involved in epithelial-mesenchymal transition, was examined using the luciferase reporter assay, western blotting, and migration and invasion assays. The association between the decreased miR-30a levels and breast cancer progression was examined in a survival analysis. miR-30a negatively regulated vimentin expression by binding to the 3'-untranslated region of Vim. Overexpression of miR-30a suppressed the migration and invasiveness phenotypes of breast cancer cell lines. Moreover, reduced tumor expression of miR-30a in breast cancer patients was associated with an unfavorable outcome, including late tumor stage, lymph node metastasis, and worse progression (mortality and recurrence) (p < 0.05). In conclusion, these findings suggest a role for miR-30a in inhibiting breast tumor invasiveness and metastasis. The finding that miR-30a downmodulates vimentin expression might provide a therapeutic target for the treatment of breast cancer.

Prognostic significance of cyclin D1, ß-catenin, and MTA1 in patients with invasive ductal carcinoma of the breast.

BACKGROUND: To investigate markers for predicting breast cancer progression, we performed a candidate gene-based study that assessed expression change of three genes, cyclin D1, ß-catenin, and metastasis-associated protein-1 (MTA1), involving in aggressive phenotypes of cancerous cells, namely hyperproliferation, epithelial-mesenchymal transition, and global transcriptional regulation. METHODS: Specimens were from 150 enrolled female patients, with invasive ductal carcinoma, followed up for more than 10 years. mRNA expression of cyclin D1, ß-catenin, and MTA1 in cancerous and noncancerous cells microdissected from the primary tumor site was determined by quantitative real-time PCR. The relationship between mRNA expression levels of the genes and clinicopathologic features was assessed by statistical analysis. Disease-free and overall survival (DFS and OS) were analyzed by Kaplan-Meier analysis with log-rank test and a multivariate Cox regression model. RESULTS: Cyclin D1 was shown to be overexpressed in late-stage breast cancer (stage III/IV). Breast cancer with lymph node metastasis (LNM) showed significantly higher frequency of overexpressed cyclin D1, ß-catenin, and MTA1 (P < 0.05). Patients carrying greater numbers of overexpressed genes had joint effects on increased risk in tumors of advanced stages (P ( trend ) = 0.03) and LNM (P ( trend ) < 0.01). In the LNM-negative group, patients whose tumors with greater number of cyclin D1, ß-catenin, and MTA1 overexpressions were associated with poorer clinical outcomes, with hazard ratio of 14.79 for OS (P = 0.015) and 7.54 for DFS (P = 0.015) using multivariate Cox regression analysis during the 10-year follow-up. CONCLUSIONS: Higher expression of cyclin D1, ß-catenin, and MTA1 mRNAs in breast cancers may prove effective in predicting unfavorable outcomes of breast cancer.

Genetic variation in the genome-wide predicted estrogen response element-related sequences is associated with breast cancer development.

INTRODUCTION: Estrogen forms a complex with the estrogen receptor (ER) that binds to estrogen response elements (EREs) in the promoter region of estrogen-responsive genes, regulates their transcription, and consequently mediates physiological or tumorigenic effects. Thus, sequence variants in EREs have the potential to affect the estrogen-ER-ERE interaction. In this study, we examined the hypothesis that genetic variations of EREs are associated with breast cancer development. METHODS: This case-control study involved 815 patients of Asian descent with incident breast cancer and 821 healthy female controls. A total of 13,737 ERE sites in the whole genome predicted by a genome-wide computational algorithm were blasted with single-nucleotide polymorphism (SNP) sequences. Twenty-one SNPs located within 2,000 bp upstream or within introns 1 and 2 of putative genes and with a minor allele frequency greater than 5% were identified and genotyped. Frequencies of SNPs were compared between cases and controls to identify SNPs associated with cancer susceptibility. RESULTS: A significant combined effect of rs12539530, an ERE SNP in intron 2 of NRCAM which codes for a cell adhesion molecule, and SNPs of ESR1, the gene coding for ER, on breast cancer risk was found. Interestingly, this combined effect was more significant in women who had experienced a longer period of lifetime estrogen exposure, supporting a hormonal etiology of this SNP in breast tumorigenesis. CONCLUSIONS: Our findings provide support for a role of genetic variation in ERE-ESR1 in determining susceptibility of breast cancer development.

Synergistic effects of polymorphisms in DNA repair genes and endogenous estrogen exposure on female breast cancer risk.

BACKGROUND: Endogenous estrogen is suggested to initiate cell proliferation and cause oxidative DNA damage during breast tumorigenesis. Cells eliminate DNA damage by means of repair enzymes. Genotypic variants of DNA damage repair genes, participating in base excision repair (BER) and nucleotide excision repair (NER) pathways, may act as modifiers that affect the association between estrogen exposure and breast cancer. METHODS: In a hospital-based case-control study of female breast cancer, DNA samples were obtained from 401 cases and 533 enrolled healthy controls, all of whom were Chinese women in Taiwan. Genotyping of polymorphisms of XRCC1 (Arg194Trp and Arg399Gln), OGG1 (Ser326Cys and Arg229Gln), ERCC2 Lys751Gln, ERCC4 Ser662Pro, and ERCC5 His1104Asp was performed and used to evaluate breast cancer susceptibility. RESULTS: Of the nonsynonymous polymorphisms, the ERCC5 1104Asp variant was significantly associated with breast cancer (odds ratio = 1.42; 95% confidence interval = 1.08-1.97), and this association was more pronounced in women with lengthy estrogen exposure. A trend toward an increased risk of developing breast cancer was observed in women who carried greater numbers of combined high-risk genotypes of BER and NER genes (P(trend) = .038). The synergistic effect of multiple genes on the increase of risk was significant in women with a longer period of estrogen exposure (>26 years), greater age at first full-term pregnancy (>26 years), a longer menarche-to-first full-term pregnancy interval (>11 years), and higher body mass index (>22) (all P<.05). CONCLUSIONS: This study demonstrates that genotype polymorphisms related to DNA damage repair confer greater susceptibility to endogenous estrogen in the development of breast cancer in women.

Genetic variants of BLM interact with RAD51 to increase breast cancer susceptibility.

The role of the familial breast cancer susceptibility genes, BRCA1 and BRCA2, in the homologous recombination (HR) pathway for DNA double-strand break (DSB) repair suggests that the mechanisms involved in HR and DNA DSB repair are of etiological importance during breast tumorigenesis. Bloom (BLM) helicase directly interacts with RAD51 recombinase, which is involved in regulating HR, and it is thus of particular interest to examine whether this interaction is associated with breast cancer susceptibility. This single-nucleotide polymorphism (SNP)-based case-control study was performed to examine this hypothesis using specimens from 933 patients with breast cancer and 1539 healthy controls. The results showed that one SNP (rs2380165) in BLM and two (rs2412546 and rs4417527) in RAD51 were associated with breast cancer risk. Furthermore, haplotype and diplotype analyses based on combinations of five SNPs in RAD51 revealed a strong association between RAD51 polymorphisms and breast cancer risk (P < 0.05). Support for the interaction between BLM and RAD51 in determining breast cancer risk came from the finding that the association between cancer risk and at-risk genotypes/haplotype pairs of RAD51 was stronger and more significant in women harboring homozygous variant alleles of BLM (P for interaction < 0.05). Interestingly, not only the intronic SNP located within the region encoding the helicase domain of BLM but also those within the RAD51-interaction domain-encoding region showed an interaction with RAD51 polymorphisms in determining breast cancer susceptibility. Our results suggest a contribution of BLM and RAD51 to breast cancer development and provide support for the tumorigenic significance of the functional interaction between these two HR proteins.

Genetic susceptibility to the development and progression of breast cancer associated with polymorphism of cell cycle and ubiquitin ligase genes.

Tumor levels of the cell cycle regulators cyclin E and p27 correlate strongly with survival in breast cancer patients and are specifically regulated by the ubiquitin ligases hCDC4 and SKP2. This study was to explore whether genetic susceptibility to breast cancer is associated with polymorphism of these genes and whether gene-gene and gene-risk factor [i.e. full-term pregnancy (FTP)] interactions are important in determining cancer risk. A two-stage case-control study based on single-nucleotide polymorphisms was performed. The first study (560 cases and 1122 controls) was to define the contribution of cell cycle and ubiquitin ligase genes to cancer susceptibility. The second study (926 cases and 923 controls) was to confirm the association identified in the first stage and to map the variant alleles. Increased breast cancer risk was associated with both polymorphism of hCDC4 and a joint effect of cyclin E and hCDC4. These associations were more significant in nulliparous women, and cancer risk associated with a lower number of FTPs was only seen in women with a higher number of high-risk genotypes, providing support for an effect of gene-risk factor interaction in determining susceptibility. Sequence variants of intron 2 in hCDC4 were found to be the most significant polymorphism and high-stage estrogen receptor (ER)-negative patients carrying the homozygous variant genotype manifested significantly poorer survival. This study concludes that polymorphism of hCDC4 is a risk factor for breast cancer development by interacting with either cyclin E or FTP and may also prove useful in predicting progression of patients with high-stage and ER-negative breast cancers.

Genetic variation in the premature aging gene WRN: a case-control study on breast cancer susceptibility.

The high risk of developing cancer seen in human genetic diseases that resemble accelerated aging provides support for a tumorigenic contribution of the mechanisms and genes responsible for regulating life span and aging. We therefore speculated that the WRN gene (encoding RECQL2, a DNA helicase), the germline mutation of which causes the progeroid disorder Werner syndrome, may be associated with breast tumorigenesis. This hypothesis was tested in this case-control study of 935 primary breast cancer patients and 1,545 healthy controls by examining single-nucleotide polymorphisms (SNPs) in WRN. We were also interested in knowing whether any identified association between WRN and breast cancer was modified by reproductive risk factors reflecting susceptibility to estrogen exposure. Our hypothesis is that because estrogen is known to promote breast cancer development via its mitogenic effect leading to cell proliferation, and because WRN is an essential gene, as its suboptimal function leads to a severe decrease in proliferation, estrogen stimulation may have a protective effect on cells harboring variant WRN, allowing them to survive and proliferate for the prolonged period needed for tumor formation. Support for this hypothesis came from the following observations: (a) one SNP in WRN was significantly associated with breast cancer risk (P = 0.002); (b) haplotype and diplotype analyses, based on different combinations of multiple SNPs in WRN, revealed a strong association with breast cancer risk; (c) this association between risk and putative high-risk genotypes was stronger and more significant in women with a longer interval between menarche and first full-term pregnancy; and (d) the protective effect conferred by having a higher number of full-term pregnancy was only significant in women with homozygous or heterozygous wild-type WRN genotypes. This study provides support for the tumorigenic role of WRN in breast cancer development, suggesting that breast cancer can be driven by the aging associated with variant WRN, the tumorigenic contribution of which might be enhanced as a result of increased cell growth due to estrogen exposure.

Diverse associations between ESR1 polymorphism and breast cancer development and progression.

PURPOSE: To test the hypothesis that polymorphisms of ESR1, the gene encoding estrogen receptor alpha (ERalpha), are associated with susceptibility, clinical phenotype, and progression of breast cancer. PATIENTS AND METHODS: A case-control study was done on 940 patients with incident breast cancer and 1,547 healthy female controls. Fifteen single-nucleotide polymorphisms (SNP) selected from chr6:152,170,379-152,466,100 (exons 1-8 of the ESR1 gene, excluding flanking sequences), reflecting major polymorphisms of this gene, were genotyped. Frequencies of SNPs were compared between cases and controls to identify SNPs associated with cancer susceptibility and between cases with different clinical phenotypes to determine the role of ESR1 polymorphism in cancer progression. RESULTS: SNPs located in one cluster in intron 1 and one haplotype, based on these SNPs, showed a significant association with breast cancer susceptibility. The tumorigenic contribution of these intron 1 SNPs was more obvious in combination with reproductive risk factors (P for interaction <0.05). One of these intron 1 SNPs was also significantly associated with low ERalpha expression in tumors. Interestingly, the same intron 1 SNPs showed a correlation with worse clinical phenotypes, including poor differentiation of tumor cells and a late stage. These intron 1 SNPs also showed a significant association with the 5-year breast cancer-specific survival rate of patients, but had opposite effects in ERalpha-negative and ERalpha-positive early-stage patients. CONCLUSIONS: Our findings provide support for diverse roles of ESR1 polymorphism in determining susceptibility in different stages of breast cancer. The differences between the important ESR1 SNPs identified in Chinese women in this study and those identified in studies on Western women with breast cancer suggest different roles of ERalpha in these two populations.

Model of human aging: recent findings on Werner's and Hutchinson-Gilford progeria syndromes.

The molecular mechanisms involved in human aging are complicated. Two progeria syndromes, Werner's syndrome (WS) and Hutchinson-Gilford progeria syndrome (HGPS), characterized by clinical features mimicking physiological aging at an early age, provide insights into the mechanisms of natural aging. Based on recent findings on WS and HGPS, we suggest a model of human aging. Human aging can be triggered by two main mechanisms, telomere shortening and DNA damage. In telomere-dependent aging, telomere shortening and dysfunction may lead to DNA damage responses which induce cellular senescence. In DNA damage-initiated aging, DNA damage accumulates, along with DNA repair deficiencies, resulting in genomic instability and accelerated cellular senescence. In addition, aging due to both mechanisms (DNA damage and telomere shortening) is strongly dependent on p53 status. These two mechanisms can also act cooperatively to increase the overall level ofgenomic instability, triggering the onset of human aging phenotypes.

DNA double-strand break repair capacity and risk of breast cancer.

A tumorigenic role of the non-homologous end-joining (NHEJ) pathway for the repair of DNA double-strand breaks (DSBs) has been suggested by our finding of a significant association between increased breast cancer risk and a cooperative effect of single-nucleotide polymorphisms in NHEJ genes. To confirm this finding, this case-control study detected both in vivo and in vitro DNA end-joining (EJ) capacities in Epstein-Barr virus-immortalized peripheral blood mononuclear cells (PBMCs) of 112 breast cancer patients and 108 healthy controls to identify individual differences in EJ capacity to repair DSB as a risk factor predisposing women to breast cancer. PBMCs from breast cancer patients consistently showed lower values of in vivo and in vitro EJ capacities than those from healthy women (P < 0.05). Logistic regression, simultaneously considering the effect of known risk factors of breast cancer, shows that the in vitro EJ capacity above the median of control subjects was associated with nearly 3-fold increased risks for breast cancer (adjusted odds ratio, 2.98; 95% confidence interval, 1.64-5.43). Furthermore, a dose-response relationship was evident between risk for breast cancer and EJ capacity, which was analyzed as a continuous variable (every unit decrease of EJ capacity being associated with an 1.09-fold increase of breast cancer risk) and was divided into tertiles based on the EJ capacity values of the controls (P for trend < 0.01). The findings support the conclusion that NHEJ may play a role in susceptibility to breast cancer.

Expression of estrogen receptor-alpha and Ki67 in relation to pathological and molecular features in early-onset infiltrating ductal carcinoma.

Estrogen causes breast cancer by triggering proliferation via an estrogen receptor (ER)-mediated mechanism. However, paradoxically, ER alpha, one of the two known ER subtypes, and the proliferation marker, Ki67, are not usually expressed in the same breast tumor. To explore whether ER alpha-positive tumors and proliferating (Ki67-positive) tumors have different tumorigenic characteristics, we performed an immunohistochemical study on 74 early-onset infiltrating ductal carcinomas of the breast. To test this hypothesis, we examined whether ER alpha-positive and Ki67-positive tumors showed differences in (i) pathological grade, (ii) three indices of tumor grade (tubule formation, nuclear pleomorphism, and mitotic number), and (iii) expression of important proteins implicated in breast tumorigenesis (cyclin D1, ErbB2, ATM, BRCA1, Rb, p53, and p21). The results of the multigenic analysis showed that ER alpha and Ki67 were the only two important markers significantly and independently associated with tumor grade, consistent with the above hypothesis. ER alpha-positive, Ki67-negative tumors frequently displayed a low tumor grade (i.e. being well differentiated), whereas Ki67-positive, ER alpha-negative tumors were more likely to exhibit a high tumor grade. In addition, positive ER alpha expression (46 of 74 cases, 62%) correlated well with positive cyclin D1 expression (p < 0.005), less nuclear pleomorphism (p < 0.001), and a low mitotic count (p < 0.005), whereas positive Ki67 expression (36 of 74 cases, 49%) correlated with reduced BRCA1 expression (p < 0.01) and high mitotic activity (p < 0.01). These findings suggest that the expressions of ER alpha and Ki67 might be involved in distinct pathological and molecular features during breast cancer development.