DNA methylation modifies the association between obesity and survival after breast cancer diagnosis

Mechanisms underlying the poor breast cancer prognosis among obese women are unresolved. DNA methylation levels are linked to obesity and to breast cancer survival. We hypothesized that obesity may work in conjunction with the epigenome to alter prognosis. Using a population-based sample of women diagnosed with first primary breast cancer, we examined modification of the obesity-mortality association by DNA methylation. In-person interviews were conducted approximately 3 months after diagnosis. Weight and height were assessed [to estimate body mass index (BMI)], and blood samples collected. Promoter methylation of 13 breast cancer-related genes was assessed in archived tumor by methylation-specific PCR and Methyl Light. Global methylation in white blood cell DNA was assessed by analysis of long interspersed elements-1 (LINE-1) and with the lumino-metric methylation assay (LUMA). Vital status among 1308 patients (with any methylation biomarker and complete BMI assessment) was determined after approximately 15 years of follow-up (N = 194/441 deaths due to breast cancer-specific/all-cause mortality). We used Cox proportional hazards regression to estimate hazard ratios (HRs) and 95 % confidence intervals (CIs) using two-sided p values of 0.05. Breast cancer-specific mortality was higher among obese (BMI ≥ 30) patients with promoter methylation in APC (HR = 2.47; 95 % CI = 1.43–4.27) and TWIST1 (HR = 4.25; 95 % CI = 1.43–12.70) in breast cancer tissue. Estimates were similar, but less pronounced, for all-cause mortality. Increased all-cause (HR =1.81; 95 % CI = 1.19–2.74) and breast cancer-specific (HR = 2.61; 95 % CI = 1.45–4.69) mortality was observed among obese patients with the lowest LUMA levels. The poor breast cancer prognosis associated with obesity may depend on methylation profiles, which warrants further investigation

Gene promoter methylation is associated with increased mortality among women with breast cancer

To better understand breast cancer etiology and progression, we explored the association between promoter methylation status of three breast cancer related genes (BRCA1, APC and p16) and survival in a large cohort of women with breast cancer. About 800 archived tumor tissues were collected from women diagnosed with a first primary invasive or in situ breast cancer in 1996–1997. The vital status of the participants was followed through the end of year 2005 with a mean follow up time of 8.0 years. Promoter methylation was assessed by methylation-specific PCR (for BRCA1) and MethyLight (for APC and p16). The association of promoter methylation and breast cancer mortality was evaluated by Cox-proportional hazards models. Methylated promoters were found in 59.0%, 48.4% and 3.6% of the tumor samples for BRCA1, APC and p16, respectively. Breast cancer-specific mortality was strongly associated with promoter methylation of p16 [HR and 95% CI: 3.53(1.83–6.78)], whereas the associations with of BRCA1 and APC were less pronounced [HR and 95% CI: 1.81(1.18–2.78) and 1.46(0.98–2.17), respectively]. Similar associations were observed with all-cause mortality. As the number of methylated genes increased, the risk of breast cancer-specific mortality also increased in a dose-dependent manner (p, trend=0.01). Importantly, even with our results stratified by hormone receptor status, promoter methylation of the three genes remained predictive of mortality. Our results suggest that promoter methylation could be promising epigenetic markers to be considered for breast cancer survival

BRCA1 promoter methylation is associated with increased mortality among women with breast cancer

Promoter-CpG island hypermethylation is a common molecular defect in cancer cells. It has been proposed as an alternative mechanism to inactivate BRCA1in the breast where somatic mutations of BRCA1 are rare. To better understand breast cancer etiology and progression, we explored the association between BRCA1 promoter methylation status and prognostic factors as well as survival among women with breast cancer. We also examined whether dietary methyl content and functional polymorphisms of genes involved in one-carbon metabolism influenced the methylation pattern. Promoter methylation of BRCA1 was assessed in 851 archived tumor tissues collected from a population-based study of women diagnosed with invasive or in situ breast cancer in 1996–1997, and who were followed for vital status through the end of 2002. About 59% of the tumors were methylated at the promoter of BRCA1. The BRCA1 promoter methylation was more frequent in invasive cancers (p=0.02) and among premenopausal cases (p=0.05). BRCA1 promoter methylation was associated with increased risk of breast cancer-specific mortality (age-adjusted HR 1.71; 95% CI: 1.05–2.78) and all-cause mortality (age-adjusted HR 1.49; 95% CI: 1.02–2.18). Among dietary methyl intakes in the year prior to the baseline interview examined, cases with lowest quintile of choline intake (<20%) had higher BRCA1 methylation level in the tumor compared to the rest (66.1% vs. 57.7%, p=0.04). Functional polymorphisms in one-carbon metabolism were not correlated with BRCA1 methylation status. Our study is the first epidemiological investigation on the prognostic value of BRCA1 promoter methylation in a large population-based cohort of breast cancer patients. Our results indicate that BRCA1 promoter methylation is an important factor to consider in predicting breast cancer survival

Prognostic significance of gene-specific promoter hypermethylation in breast cancer patients

The association between promoter methylation status and survival was investigated in a large cohort of women with breast cancer, participants in the Long Island Breast Cancer Study Project. Archived tumor tissues (n=839) were collected from women diagnosed with a first primary invasive or in situ breast cancer in 1996-1997. Vital status was followed through the end of 2005 with a mean follow up time of 8 years. Promoter methylation of 8 breast cancer-related genes was assessed by MethyLight. The frequencies of methylation for HIN1, RASSF1A, DAPK1, GSTP1, CyclinD2, TWIST, CDH1 and RARβ were 62.9%, 85.2%, 14.1%, 27.8%, 19.6%, 15.3%, 5.8% and 27.6%, respectively. Since survival rates of in situ and invasive breast cancers are substantially different, survival analyses were conducted within 670 invasive cases with complete data on all genes. Age-adjusted Cox-proportional hazards models revealed that GSTP1, TWIST and RARβ methylation was significantly associated with higher breast cancer-specific mortality. Methylation of GSTP1 and RARβ were significantly associated with higher all-cause mortality. To investigate the relationship between the number of methylated genes and breast cancer-specific mortality, we included previously published MethyLight data on p16 and APC methylation status. Breast cancer-specific mortality increased in a dose-dependent manner with increasing number of methylated genes (Ptrend = 0.002), although confidence intervals were wide. Our results suggest that promoter methylation, particularly for a panel of genes, has the potential to be used as a biomarker for predicting prognosis in breast cancer

Environmental toxins and breast cancer on Long Island. I. Polycyclic aromatic hydrocarbon DNA adducts

Polycyclic aromatic hydrocarbons (PAH) are potent mammary carcinogens in rodents, but their effect on breast cancer development in women is not clear. To examine whether currently measurable PAH damage to DNA increases breast cancer risk, a population-based case-control study was undertaken on Long Island, NY. Cases were women newly diagnosed with in situ and invasive breast cancer; controls were randomly selected women frequency matched to the age distribution of cases. Blood samples were donated by 1102 (73.0%) and 1141 (73.3%) of case and control respondents, respectively. Samples from 576 cases and 427 controls were assayed for PAH-DNA adducts using an ELISA. The geometric mean (and geometric SD) of the log-transformed levels of PAH-DNA adducts on a natural scale was slightly, but nonsignificantly, higher among cases [7.36 (7.29)] than among controls [6.21 (4.17); p = 0.51]. The age-adjusted odds ratio (OR) for breast cancer in relation to the highest quintile of adduct levels compared with the lowest was 1.51 [95% confidence interval (CI), 1.04 –2.20], with little or no evidence of substantial confounding (corresponding multivariate-adjusted OR, 1.49; 95% CI, 1.00 –2.21). There was no consistent elevation in risk with increasing adduct levels, nor was there a consistent association between adduct levels and two of the main sources of PAH, active or passive cigarette smoking or consumption of grilled and smoked foods. These data indicate that PAH-DNA adduct formation may influence breast cancer development, although the association does not appear to be dose dependent and may have a threshold effect

The influence of one-carbon metabolism on gene promoter methylation in a population-based breast cancer study

Abnormal methylation in gene promoters is a hallmark of the cancer genome; however, factors that may influence promoter methylation have not been well elucidated. As the one-carbon metabolism pathway provides the universal methyl donor for methylation reactions, perturbation of this pathway might influence DNA methylation and, ultimately, affect gene functions. Utilizing approximately 800 breast cancer tumor tissues from a large population-based study, we investigated the relationships between dietary and genetic factors involved in the one-carbon metabolism pathway and promoter methylation of a panel of 13 breast cancer-related genes. We found that CCND2, HIN1 and CHD1 were the most “dietary sensitive” genes, as methylation of their promoters was associated with intakes of at least two out of the eight dietary methyl factors examined. On the other hand, some micronutrients (i.e., B2 and B6) were more “epigenetically active” as their intake levels correlated with promoter methylation status in 3 out of the 13 breast cancer genes evaluated. Both positive (hypermethylation) and inverse (hypomethylation) associations with high micronutrient intake were observed. Unlike what we saw for dietary factors, we did not observe any clear patterns between one-carbon genetic polymorphisms and the promoter methylation status of the genes examined. Our results provide preliminary evidence that one-carbon metabolism may have the capacity to influence the breast cancer epigenome. Given that epigenetic alterations are thought to occur early in cancer development and are potentially reversible, dietary modifications may offer promising venues for cancer intervention and prevention

Global DNA Methylation, Measured by the Luminometric Methylation Assay (LUMA), Associates with Postmenopausal Breast Cancer in Non-Obese and Physically Active Women

Introduction: Little is known about how modifiable lifestyle factors interact with the epigenome to influence disease. Body mass index (BMI, weight kg/height m2) and physical activity are associated with postmenopausal breast cancer, but the mechanisms are not well-understood. We hypothesized that BMI or physical activity may modify the association between markers of global DNA methylation and postmenopausal breast cancer risk

Mutations in p53, p53 protein overexpression and breast cancer survival

p53 is an important tumor-suppressor gene that encodes p53 protein, a molecule involved in cell cycle regulation, and has been inconsistently linked to breast cancer survival. Using archived tumor tissue from a population-based sample of 859 women diagnosed with breast cancer between 1996–1997, we determined p53 mutations in exons 5–8 and p53 protein overexpression. We examined the association of p53 mutations with overexpression and selected tumor clinical parameters. We assessed whether either p53 marker was associated with survival through 2002, adjusting for other tumor markers and prognostic factors. The prevalence of protein overexpression in the tumor was 36% (307/859) and any p53 mutation was 15% (128/859). p53 overexpression was positively associated with the presence of any p53 mutation (odds ratio (OR)=2.2, 95% confidence interval (CI)=1.5–3.2), particularly missense mutations (OR=7.0, 95%CI=3.6–13.7). Negative estrogen and progesterone receptor status (ER/PR) was positively associated with both p53 protein overexpression (OR = 2.6, 95% CI = 1.7–4.0), and p53 mutation (OR = 3.9, 95% CI = 2.4–6.5). Any p53 mutation and missense mutations, but not p53 protein overexpression, were associated with breast cancer-specific mortality (Hazard ratio HR=1.7, 95%CI=1.0–2.8; HR=2.0, 95%CI=1.1–3.6, respectively) and all-cause mortality (HR=1.5, 95%CI=1.0–2.4; HR=2.0, 95%CI=1.2–3.4, respectively); nonsense mutations were associated only with breast cancer-specific mortality (HR=3.0, 95%CI=1.1–8.1). These associations however did not remain after adjusting for ER/PR status. Thus, in this population-based cohort of women with breast cancer, although p53 protein overexpression and p53 mutations were associated with each other, neither independently impacted breast-cancer specific or all-causing mortality after considering ER/PR status

Associations between Polycyclic Aromatic Hydrocarbon–Related Exposures and p53 Mutations in Breast Tumors

Background: Previous studies have suggested that polycyclic aromatic hydrocarbons (PAHs) may be associated with breast cancer. However, the carcinogenicity of PAHs on the human breast remains unclear. Certain carcinogens may be associated with specific mutation patterns in the p53 tumor suppressor gene, thereby contributing information about disease etiology. Objectives: We hypothesized that associations of PAH-related exposures with breast cancer would differ according to tumor p53 mutation status, effect, type, and number. Methods: We examined this possibility in a population-based case–control study using polytomous logistic regression. As previously reported, 151 p53 mutations among 859 tumors were identified using Surveyor nuclease and confirmed by sequencing. Results: We found that participants with p53 mutations were less likely to be exposed to PAHs (assessed by smoking status in 859 cases and 1,556 controls, grilled/smoked meat intake in 822 cases and 1,475 controls, and PAH–DNA adducts in peripheral mononuclear cells in 487 cases and 941 controls) than participants without p53 mutations. For example, active and passive smoking was associated with p53 mutation–negative [odds ratio (OR) = 1.55; 95% confidence interval (CI), 1.11–2.15] but not p53 mutation–positive (OR = 0.77; 95% CI, 0.43–1.38) cancer (ratio of the ORs = 0.50, p < 0.05). However, frameshift mutations, mutation number, G:C→A:T transitions at CpG sites, and insertions/deletions were consistently elevated among exposed subjects. Conclusions: These findings suggest that PAHs may be associated with specific breast tumor p53 mutation subgroups rather than with overall p53 mutations and may also be related to breast cancer through mechanisms other than p53 mutation

Associations between Polycyclic Aromatic Hydrocarbon–Related Exposures and p53 Mutations in Breast Tumors

BackgroundPrevious studies have suggested that polycyclic aromatic hydrocarbons (PAHs) may be associated with breast cancer. However, the carcinogenicity of PAHs on the human breast remains unclear. Certain carcinogens may be associated with specific mutation patterns in the p53 tumor suppressor gene, thereby contributing information about disease etiology.ObjectivesWe hypothesized that associations of PAH-related exposures with breast cancer would differ according to tumor p53 mutation status, effect, type, and number.MethodsWe examined this possibility in a population-based case–control study using polytomous logistic regression. As previously reported, 151 p53 mutations among 859 tumors were identified using Surveyor nuclease and confirmed by sequencing.ResultsWe found that participants with p53 mutations were less likely to be exposed to PAHs (assessed by smoking status in 859 cases and 1,556 controls, grilled/smoked meat intake in 822 cases and 1,475 controls, and PAH–DNA adducts in peripheral mononuclear cells in 487 cases and 941 controls) than participants without p53 mutations. For example, active and passive smoking was associated with p53 mutation–negative [odds ratio (OR) = 1.55; 95% confidence interval (CI), 1.11–2.15] but not p53 mutation–positive (OR = 0.77; 95% CI, 0.43–1.38) cancer (ratio of the ORs = 0.50, p < 0.05). However, frameshift mutations, mutation number, G:C→A:T transitions at CpG sites, and insertions/deletions were consistently elevated among exposed subjects.ConclusionsThese findings suggest that PAHs may be associated with specific breast tumor p53 mutation subgroups rather than with overall p53 mutations and may also be related to breast cancer through mechanisms other than p53 mutation