In vitro model for DNA double-strand break repair analysis in breast cancer reveals cell type-specific associations with age and prognosis.

Dysfunction of homologous recombination is a common denominator of changes associated with breast cancer-predisposing mutations. In our previous work, we identified a functional signature in peripheral blood lymphocytes from women who were predisposed that indicated a shift from homologous recombination to alternative, error-prone DNA double-strand break (DSB) repair pathways. To capture both hereditary and nonhereditary factors, we newly established a protocol for isolation and ex vivo analysis of epithelial cells, epithelial-mesenchymal transition cells (EMTs), and fibroblasts from breast cancer specimens (147 patients). By applying a fluorescence-based test system, we analyzed the error-prone DSB repair pathway microhomology-mediated end joining in these tumor-derived cell types and peripheral blood lymphocytes. In parallel, we investigated DNA lesion processing by quantitative immunofluorescence microscopy of histone H2AX phosphorylated on Ser139 focus after radiomimetic treatment. Our study reveals elevated histone H2AX phosphorylated on Ser139 damage removal in epithelial cells, not EMTs, and poly(ADP-ribose)polymerase inhibitor sensitivities, which suggested a DSB repair pathway shift with increasing patient age. Of interest, we found elevated microhomology-mediated end joining in EMTs, not epithelial cells, from patients who received a treatment recommendation of adjuvant chemotherapy, that is, those with high-risk tumors. Our discoveries of altered DSB repair activities in cells may serve as a method to further classify breast cancer to predict responsiveness to adjuvant chemotherapy and/or therapeutics that target DSB repair-dysfunctional tumors.-Deniz, M., Kaufmann, J., Stahl, A., Gundelach, T., Janni, W., Hoffmann, I., Keimling, M., Hampp, S., Ihle, M., Wiesmüller, L. In vitro model for DNA double-strand break repair analysis in breast cancer reveals cell type-specific associations with age and prognosis.

A systematic review and meta-analysis of physical activity and endometrial cancer risk.

Physical activity is related to decreased endometrial cancer risk. However, a comprehensive investigation of activity domains, intensities, time periods in life, and potential interaction with body mass index is unavailable. We performed a meta-analysis of physical activity and endometrial cancer studies published through October 2014. We identified 33 eligible studies comprising 19,558 endometrial cancer cases. High versus low physical activity was related to reduced endometrial cancer risk [relative risk (RR) = 0.80; 95% confidence interval (CI) 0.75-0.85]. The corresponding RRs for recreational activity, occupational activity, household activity, and walking were 0.84 (95% CI 0.78-0.91), 0.81 (95% CI 0.75-0.87), 0.70 (95% CI 0.47-1.02), and 0.82 (95% CI 0.69-0.97), respectively (Pdifference). Walking/biking for transportation, walking for recreation, and walking without specification revealed summary RRs of 0.70 (95% CI 0.58-0.85), 0.94 (95% CI 0.76-1.17), and 0.88 (95% CI 0.52-1.50), respectively (Pdifference). Inverse associations were noted for light (RR 0.65; 95% CI 0.49-0.86), moderate to vigorous (RR 0.83; 95 % CI 0.71-0.96), and vigorous activity (RR 0.80; 95% CI 0.72-0.90; (Pdifference). A statistically significant inverse relation was found for postmenopausal (RR 0.81; 95% CI 0.67-0.97), but not premenopausal women (RR 0.74; 95% CI 0.49-1.13; (Pdifference). Physical activity performed during childhood/adolescence, young adulthood/midlife, and older age yielded RRs of 0.94 (95% CI 0.82-1.08), 0.77 (95% CI 0.58-1.01), and 0.69 (95% CI 0.37-1.28), respectively (Pdifference). An inverse relation was evident in overweight/obese (RR 0.69; 95% CI 0.52-0.91), but not normal weight women (RR 0.97; 95% CI 0.84-1.13; (Pdifference). In conclusion, recreational physical activity, occupational physical activity, and walking/biking for transportation are related to decreased endometrial cancer risk. Inverse associations are evident for physical activity of light, moderate to vigorous, and vigorous intensities. The inverse relation with physical activity is limited to women who are overweight or obese.

Physical activity and risk of pancreatic cancer: a systematic review and meta-analysis.

Physical activity may prevent pancreatic cancer by regulating body weight and decreasing insulin resistance, DNA damage, and chronic inflammation. Previous meta-analyses found inconsistent evidence for a protective effect of physical activity on pancreatic cancer but those studies did not investigate whether the association between physical activity and pancreatic cancer varies by smoking status, body mass index (BMI), or level of consistency of physical activity over time. To address these issues, we conducted an updated meta-analysis following the PRISMA guidelines among 30 distinct studies with a total of 10,501 pancreatic cancer cases. Random effects meta-analysis of cohort studies revealed a weak, statistically significant reduction in pancreatic cancer risk for high versus low levels of physical activity (relative risk (RR) 0.93, 95 % confidence interval (CI) 0.88-0.98). By comparison, case-control studies yielded a stronger, statistically significant risk reduction (RR 0.78, 95 % CI 0.66-0.94; p-difference by study design = 0.07). When focusing on cohort studies, physical activity summary risk estimates appeared to be more pronounced for consistent physical activity over time (RR 0.86, 95 % CI 0.76-0.97) than for recent past physical activity (RR 0.95, 95 % CI 0.90-1.01) or distant past physical activity (RR 0.95, 95 % CI 0.79-1.15, p-difference by timing in life of physical activity = 0.36). Physical activity summary risk estimates did not differ by smoking status or BMI. In conclusion, physical activity is not strongly associated with pancreatic cancer risk, and the relation is not modified by smoking status or BMI level. While overall findings were weak, we did find some suggestion of potential pancreatic cancer risk reduction with consistent physical activity over time.

The association between physical activity and gastroesophageal cancer: systematic review and meta-analysis.

Physical activity may decrease gastroesophageal cancer risk through a reduction of oxidative stress and decreased chronic inflammation, yet few epidemiologic studies have been able to report a clear inverse association between physical activity and gastroesophageal cancer. Because no meta-analysis has investigated the relation of physical activity to gastroesophageal cancer, we conducted a comprehensive systematic review and meta-analysis according to the PRISMA guidelines based on 24 studies with a total of 15,745 cases. When we compared high versus low physical activity levels and summarized associations according to anatomic site and tumor histology, risk reductions were evident for esophageal adenocarcinoma [relative risk (RR) = 0.79, 95% confidence interval (CI) = 0.66-0.94], gastric cardia adenocarcinoma (RR = 0.83, 95% CI = 0.69-0.99) and gastric non-cardia adenocarcinoma (RR = 0.72, 95% CI = 0.62-0.84). The risk reduction for esophageal squamous cell carcinoma (RR = 0.94, 95% CI = 0.41-2.16) became statistically significant (RR = 0.66, 95% CI = 0.46-0.96) after excluding an influential study. The test for heterogeneity by gastroesophageal cancer subtype was statistically non-significant (p-difference = 0.71). The RR of total gastroesophageal cancer for high versus low physical activity was 0.82 (95% CI = 0.74-0.90). A dose-response analysis of frequency of physical activity and total gastroesophageal cancer risk revealed that the greatest risk reduction was achieved among those engaging in moderate to vigorous physical activity five times per week (RR = 0.67, 95% CI = 0.58-0.79). Our results provide support for an inverse relation of physical activity, in particular exercise frequency, to gastroesophageal cancer risk.

Physical activity in relation to risk of hematologic cancers: a systematic review and meta-analysis.

BACKGROUND: Despite the existence of numerous biologic pathways potentially linking increased physical activity to decreased risk of hematologic cancers, the associations between physical activity and subtype-specific hematologic cancers have not been comprehensively quantified. METHODS: We conducted a systematic review and meta-analysis of physical activity in relation to subtype-specific hematologic cancers. We summarized the data from 23 eligible studies (15 cohort and eight case-control studies) and estimated summary relative risks (RR) and 95% confidence intervals (CI) using random-effects models. RESULTS: When comparing high versus low physical activity levels, the RR for non-Hodgkin lymphoma was 0.91 (95% CI, 0.82-1.00), for Hodgkin lymphoma it was 0.86 (95% CI, 0.58-1.26), for leukemia it was 0.97 (95% CI, 0.84-1.13), and for multiple myeloma it was 0.86 (95% CI, 0.68-1.09). When focusing on subtypes of non-Hodgkin lymphoma, the RR for diffuse large B-cell lymphoma was 0.95 (95% CI, 0.80-1.14) and for follicular lymphoma it was 1.01 (95% CI, 0.83-1.22). In an exploratory analysis combining all hematologic cancers, high versus low physical activity levels yielded a statistically significant RR of 0.93 (95% CI, 0.88-0.99). CONCLUSIONS: Physical activity showed statistically nonsignificant associations with risks of non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma, and leukemia. These findings may not represent a true lack of associations given the variation in high versus low physical activity definitions, the quality of physical activity assessments, and the variability in hematologic cancer classification schemes in individual studies. IMPACT: Physical activity is unrelated to risks of subtype-specific hematologic cancers.

Physical activity, diabetes, and risk of thyroid cancer: a systematic review and meta-analysis.

Thyroid cancer incidence has been increasing more rapidly over time than the occurrence of cancers of other sites, and interest in potential adverse relations of diabetes and lack of physical activity to thyroid cancer risk is accumulating. We conducted a systematic review and meta-analysis of published epidemiologic studies on the relations of physical activity and diabetes to thyroid cancer according to the Meta-analysis of Observational Studies in Epidemiology guidelines. Published studies were identified through a search in MEDLINE and EMBASE. Random-effects models were used to summarize thyroid cancer risk estimates comparing high versus low levels of physical activity, and separately, comparing individuals with diabetes versus those without diabetes. Meta-regression analyses were performed to evaluate potential effect modification by study design and thyroid cancer risk factors. Information was extracted from seven studies of physical activity and thyroid cancer and from six studies of diabetes and thyroid cancer. The number of individuals from studies on physical activity was 939,305 (yielding 2,250 incident thyroid cancer cases) and from studies on diabetes it was 960,840 (yielding 1,230 cases). The summary relative risk (RR) estimate from cohort and case-control studies combined indicated no association between physical activity and thyroid cancer (summary RR 1.06, 95 % confidence interval (CI) 0.79-1.42). Subgroup-analyses revealed a significant positive association between physical activity and thyroid cancer in cohort studies (summary RR 1.28; 95 % CI 1.01-1.63), whereas the relation was suggestively inverse in case-control studies (summary RR 0.70; 95 % CI 0.48-1.03; p for heterogeneity = 0.005). Individuals with diabetes showed a borderline statistically significant increased risk of thyroid cancer compared with those without diabetes (summary RR 1.17; 95 % CI 0.99-1.39). The relations of physical activity and diabetes to thyroid cancer were not modified by sex, number of adjustment factors, and adjustments for adiposity, smoking, and study quality. In this comprehensive systematic review and meta-analysis, no significant association between physical activity and thyroid cancer was found. Diabetes showed a suggestive positive relation with risk of thyroid cancer.

Comparison of associations of body mass index, abdominal adiposity, and risk of colorectal cancer in a large prospective cohort study.

BACKGROUND: Increased body mass index (BMI) is an established colorectal cancer risk factor. High waist circumference or waist-hip-ratio (WHR) may better reflect an abnormal metabolic state and be more predictive of colorectal cancer risk than BMI. METHODS: We examined BMI, waist circumference, WHR, and hip circumference in relation to colorectal cancer risk among 203,177 participants followed for 10 years. We derived standardized colorectal cancer risk estimates for each anthropometric parameter and compared predictive characteristics (Harrell's C-index). In women, we examined whether hormone replacement therapy (HRT) use modified the associations between anthropometric measures and colorectal cancer. RESULTS: We ascertained 2,869 colorectal cancers. In men, increased colon cancer risks were associated with BMI [HR per SD, 1.14; 95% confidence interval (CI), 1.08-1.20], waist circumference (HR per SD, 1.17; 95% CI, 1.08-1.27), and WHR (HR per SD, 1.09; 95% CI, 1.04-1.14). In women, anthropometric variables were unrelated to colon cancer. For men and women, anthropometric variables were unrelated to rectal cancer. Compared with BMI, waist circumference and WHR did not materially influence colon cancer prediction models [C-index changes: -0.0041 and 0.0046 (men); 0.0004 and 0.0005 (women)]. In current HRT users, colon cancer was inversely or suggestively inversely associated with waist circumference (HR per SD, 0.78; 95% CI, 0.63-0.97) and WHR (HR per SD, 0.88; 95% CI, 0.76-1.01), but positively related to hip circumference (HR per SD, 1.39; 95% CI, 1.13-1.71). CONCLUSION: BMI, waist circumference, and WHR show comparable positive associations with colon cancer in men. Associations between anthropometric measures and colon cancer are weak or null in women, but there is some evidence for effect modification by HRT. IMPACT: These findings may improve our understanding of the relation of adiposity to colorectal cancer.

The power of DNA double-strand break (DSB) repair testing to predict breast cancer susceptibility.

Most presently known breast cancer susceptibility genes have been linked to DSB repair. To identify novel markers that may serve as indicators for breast cancer risk, we performed DSB repair analyses using a case-control design. Thus, we examined 35 women with defined familial history of breast and/or ovarian cancer (first case group), 175 patients with breast cancer (second case group), and 245 healthy women without previous cancer or family history of breast cancer (control group). We analyzed DSB repair in peripheral blood lymphocytes (PBLs) by a GFP-based test system using 3 pathway-specific substrates. We found increases of microhomology-mediated nonhomologous end joining (mmNHEJ) and nonconservative single-strand annealing (SSA) in women with familial risk vs. controls (P=0.0001-0.0022) and patients with breast cancer vs. controls (P=0.0004-0.0042). Young age (<50) at initial diagnosis of breast cancer, which could be indicative of genetic predisposition, was associated with elevated SSA using two different substrates, amounting to similar odds ratios (ORs=2.54-4.46, P=0.0059-0.0095) as for familial risk (ORs=2.61-4.05, P=0.0007-0.0045). These findings and supporting validation data underscore the great potential of detecting distinct DSB repair activities in PBLs as method to estimate breast cancer susceptibility beyond limitations of genotyping and to predict responsiveness to therapeutics targeting DSB repair-dysfunctional tumors.

Functional characterization connects individual patient mutations in ataxia telangiectasia mutated (ATM) with dysfunction of specific DNA double-strand break-repair signaling pathways.

Ataxia telangiectasia mutated (ATM) has multiple functions in homologous recombination (HR) and nonhomologous end joining (NHEJ), which lead to conflicting data regarding its DNA double-strand break-repair (DSBR) functions in previous studies. To explore the effect of clinically relevant ATM mutations, we characterized DSBR between mutated EGFP genes and ATM kinase signaling in 9 lymphoblastoid cell lines (LCLs) derived from patients with ataxia telangiectasia (AT) with defined vs. 3 control LCLs without ATM mutations. Our study revealed that the DSBR phenotype in AT cells is not uniform but appears to depend on the mutation, causing up to 32-fold increased or up to 3-fold decreased activities in particular pathways. Comparison with a further 10 LCLs mutated in downstream factors (BRCA1, BRCA2, Nibrin, Rad50, and Chk2) showed that the most diametrically opposed DSBR patterns in AT cells phenocopied NBN/RAD50 or BRCA1 mutations. Notably, reexpressing wild-type ATM reversed these defects by 2.3- to 3.5-fold. Our data suggest that ATM stimulates repair proteins such as Nibrin, which execute HR, single-strand annealing (SSA), and NHEJ. Concomitantly, ATM minimizes error-prone repair (SSA and NHEJ) through activation of surveillance factors such as BRCA1. Since the outcome of the individual defect can be diametrically opposed, distinguishing repair patterns in patients with ATM mutations may also be relevant regarding therapeutic responses.

DNA double-strand break repair activities in mammary epithelial cells--influence of endogenous p53 variants.

Intriguingly, all 10 breast cancer susceptibility genes known today are directly or indirectly related to DNA double-strand break (DSB) repair suggesting a critical role of DSB repair dysfunction in the etiology of this tumor entity. We and others had previously provided evidence indicating that the breast cancer susceptibility gene product p53 controls DSB repair. Experiments with ectopically expressed proteins showed that oncogenic mutants of p53 deregulate homologous recombination (HR) and possibly also non-homologous end joining (NHEJ). Here, we systematically analyzed the role of different p53 variants endogenously expressed in a series of mammary epithelial cell lines. We provide evidence that endogenous wild-type p53 represses HR, particularly between short homologies that strengthens the idea of a quality control mechanism underlying HR regulation. To a lesser extent, p53 also downregulates microhomology-mediated NHEJ and single-strand annealing. Our data also suggest that repression of NHEJ regulation may require the extreme C-terminus, whereas the oligomerization and core domains are involved in HR regulation. We show that depending on the individual mutation, p53 mutants retain more or less partial DSB repair downregulatory activities when compared with loss of p53. All in all, relative effects on distinct DSB repair pathways and discrimination between HR substrates with perfectly versus imperfectly homologous sequences represent good markers for a p53 defect due to a specific mutation. Thus, advanced DSB repair analysis may serve as a novel assay for the functional classification of p53 mutations.

A sensitive test for the detection of specific DSB repair defects in primary cells from breast cancer specimens.

Increasing evidence indicates that breast cancer pathogenesis is linked with DNA double-strand break (DSB) repair dysfunction. This conclusion is based on advances in the study of functions of breast cancer susceptibility genes such as BRCA1 and BRCA2, on the identification of breast cancer-associated changes regarding the genetics, expression, and localization of multiple DSB repair factors, and on observations indicating enhanced radiation-induced chromosomal damage in cells from predisposed individuals and sporadic breast cancer patients. In this pilot study, we describe a sensitive method for the analysis of DSB repair functions in mammary carcinomas. Using this method we firstly document alterations in pathway-specific DSB repair activities in primary cells originating from familial as well as sporadic breast cancer. In particular, we identified increases in the mutagenic nonhomologous end joining and single-strand annealing mechanisms in sporadic breast cancers with wild-type BRCA1 and BRCA2, and, thus, similar phenotypes to tumors with mutant alleles of BRCA1 and BRCA2. This suggests that detection of error-prone DSB repair activities may be useful to extend the limits of genotypic characterization of high-risk susceptibility genes. This method may, therefore, serve as a marker for breast cancer risk assessment and, even more importantly, for the prediction of responsiveness to targeted therapies such as to inhibitors of poly(ADP-ribose)polymerase (PARP1).

Resveratrol modulates DNA double-strand break repair pathways in an ATM/ATR-p53- and -Nbs1-dependent manner.

Resveratrol (RV) inhibits tumour initiation, promotion and progression which has mainly been explained by its properties in cell cycle control and apoptosis induction. So far, ambiguous observations have been published regarding its influence on genomic stability. To study RV's effects on DNA double-strand break (DSB) repair, we applied the established enhanced green fluorescent protein (EGFP)- and I-SceI-based assay system on RV-treated lymphoblastoid cell lines (LCLs). We show that RV inhibits both, homologous recombination (HR) and non-homologous end joining (NHEJ) independently of its known growth and death regulatory functions. Using (i) the isogenic cell lines TK6 and WTK1, which differ in their p53 status, (ii) LCLs from patients with ataxia telangiectasia, (iii) shRNA-mediated p53 knockdown and (iv) chemical inhibition of ATM/ATR by caffeine, we established an ATM-p53-dependent pathway of HR inhibition by RV. Additional use of LCLs from Nijmegen breakage syndrome patients furthermore provided evidence for an ATM/ATR-Nbs1-dependent inhibition of microhomology-mediated NHEJ after RV treatment. We propose that activation of ATM and/or ATR is a central effect of RV. Repression of error-prone recombination subpathways could at least partially explain the chemopreventive effects of this natural plant constituent in animal cancer models.