Ramona G. Dumitrescu

Adipokines in Plasma and Breast Tissues: Associations with Breast Cancer Risk Factors

Background: Blood adipokines are associated with breast cancer risk; however, blood–breast adipokine correlations and factors that explain variation in adipokines are unknown. Methods: Plasma (n = 155) and breast (n = 85) leptin and adiponectin were assessed by immunoassays in women with no history of cancer. Multivariable-adjusted regression models were used to determine breast adipokine associations. Results: Through body mass index (BMI)-adjusted analyses, we initially observed positive plasma–breast correlations for leptin (r = 0.41, P = 0.0002) and adiponectin (r = 0.23, P = 0.05). The positive plasma–breast correlation for leptin was strongest among normal weight women (r = 0.62), whereas the correlation for adiponectin was strongest among obese women (r = 0.31). In multivariable models, adjusting for BMI, demographic, reproductive, and lifestyle factors, plasma leptin was not associated with breast leptin, and only the highest quartile of plasma adiponectin was associated with tissue levels. Of the risk factors investigated, those that contributed most to the variation in breast tissue adipokines were BMI and race for leptin, oral contraceptive use and smoking status for adiponectin. Conclusions: Although we report positive plasma–breast adipokine correlations overall, plasma adipokine concentrations may not be good surrogates for breast concentrations among all women. Predictors of breast adipokines vary, depending on subject characteristics, possibly explaining inconsistent epidemiologic results and they implicate differing pathways toward carcinogenesis. Impact: A clearer understanding of the relationships between plasma adipokines and their levels within the target organ is necessary to better understand the impact of these hormones on breast cancer risk. Future studies are needed to identify additional factors associated with breast adipokines in target tissues. Cancer Epidemiol Biomarkers Prev; 21(10); 1745–55. ©2012 AACR.

Racial differences in genome-wide methylation profiling and gene expression in breast tissues from healthy women

Breast cancer is more common in European Americans (EAs) than in African Americans (AAs) but mortality from breast cancer is higher among AAs. While there are racial differences in DNA methylation and gene expression in breast tumors, little is known whether such racial differences exist in breast tissues of healthy women. Genome-wide DNA methylation and gene expression profiling was performed in histologically normal breast tissues of healthy women. Linear regression models were used to identify differentially-methylated CpG sites (CpGs) between EAs (n = 61) and AAs (n = 22). Correlations for methylation and expression were assessed. Biological functions of the differentially-methylated genes were assigned using the Ingenuity Pathway Analysis. Among 485 differentially-methylated CpGs by race, 203 were hypermethylated in EAs, and 282 were hypermethylated in AAs. Promoter-related differentially-methylated CpGs were more frequently hypermethylated in EAs (52%) than AAs (27%) while gene body and intergenic CpGs were more frequently hypermethylated in AAs. The differentially-methylated CpGs were enriched for cancer-associated genes with roles in cell death and survival, cellular development, and cell-to-cell signaling. In a separate analysis for correlation in EAs and AAs, different patterns of correlation were found between EAs and AAs. The correlated genes showed different biological networks between EAs and AAs; networks were connected by Ubiquitin C. To our knowledge, this is the first comprehensive genome-wide study to identify differences in methylation and gene expression between EAs and AAs in breast tissues from healthy women. These findings may provide further insights regarding the contribution of epigenetic differences to racial disparities in breast cancer.

DNA methylation and histone modifications in breast cancer.

Breast cancer is caused by progressive genetic alterations and epigenetic changes that trigger tumor development. Among the epigenetic alterations descried in breast cancer, DNA promoter methylation has been extensively studied and observed in genes involved in several critical signaling pathways that initiate and promote breast tumorigenesis. In addition to DNA hypermethylation, global methylation levels have been observed to decrease with breast cancer progression. In close connection with DNA methylation changes, histone modifications, especially the acetylation and methylation of histone lysines, play an essential role in the nucleosomes remodeling and gene expression regulation in breast cancer.

Relationships among folate, alcohol consumption, gene variants in one-carbon metabolism and p16INK4a methylation and expression in healthy breast tissues.

p16(INK4a) is a tumor suppressor gene, frequently hypermethylated in breast cancer; this epigenetic silencing of p16(INK4a) occurs early in carcinogenesis. The risk factors and functional consequences of p16(INK4a) methylation are unknown. Alcohol consumption, a breast cancer risk factor, impedes folate metabolism and may thereby alter gene methylation since folate plays a pivotal role in DNA methylation. In a cross-sectional study of 138 women with no history of breast cancer who underwent reduction mammoplasty, we studied breast cancer risk factors, plasma and breast folate concentrations, variation in one-carbon metabolism genes, p16(INK4a) promoter methylation and P16 protein expression. Logistic regression was used to estimate multivariable-adjusted odds ratios (OR) and 95% confidence intervals (CI). p16(INK4a) methylation was negatively correlated with P16 expression (r = -0.28; P = 0.002). Alcohol consumption was associated with lower breast folate (P = 0.03), higher p16(INK4a) promoter methylation (P = 0.007) and less P16 expression (P = 0.002). Higher breast folate concentrations were associated with lower p16(INK4a) promoter methylation (P = 0.06). Genetic variation in MTRR (rs1801394) and MTHFD1 (rs1950902) was associated with higher p16 (INK4a) promoter methylation (OR = 2.66, 95% CI: 1.11-6.42 and OR = 2.72, 95% CI: 1.12-6.66, respectively), whereas variation in TYMS (rs502396) was associated with less P16 protein expression (OR = 0.22, 95% CI: 0.05-0.99). Given that this is the first study to indicate that alcohol consumption, breast folate and variation in one-carbon metabolism genes are associated with p16(INK4a) promoter methylation and P16 protein expression in healthy tissues; these findings require replication.

Alcohol and Breast Cancer Risk

Breast cancer is the most common cancer in women in the United States, other than non-melanotic skin cancer and is the second leading cause of death in women. Breast cancer is a multifactorial disease, resulting from gene-environmental interactions. Among the bestknown breast cancer risk factors, most of them cannot be controlled or modified. However, alcohol consumption is a modifiable risk factor that represents the leading non-hormonally related breast cancer risk factor. Alcohol intake is associated with increased breast cancer risk by the majority of epidemiological studies and experimental animal studies. Conflicting data has been generated about the relationship between the breast cancer risk and alcohol type, time of use and dose. Ethanol may cause breast cancer through different mechanisms, such as the induction of mutations by the acetaldehyde, changes in estrogen levels and estrogen receptor response, through oxidative damage by generating reactive oxygen species and/or the effect on one-carbon metabolism pathways on DNA methylation and DNA synthesis. Genetic variants for all these potential pathways involved in alcohol-induced breast cancers might increase a woman’s breast cancer risk. Definite studies on breast cancer etiology would lead to improved public health recommendations, allowing women to make individual choices about their lifestyle and might guide to better prevention strategies.

Associations between genetic variation in one-carbon metabolism and LINE-1 DNA methylation in histologically normal breast tissues.

Genome-wide DNA hypomethylation is an early event in the carcinogenic process. Percent methylation of long interspersed nucleotide element-1 (LINE-1) is a biomarker of genome-wide methylation and is a potential biomarker for breast cancer. Understanding factors associated with percent LINE-1 DNA methylation in histologically normal tissues could provide insight into early stages of carcinogenesis. In a cross-sectional study of 121 healthy women with no prior history of cancer who underwent reduction mammoplasty, we examined associations between plasma and breast folate, genetic variation in one-carbon metabolism, and percent LINE-1 methylation using multivariable regression models (adjusting for race, oral contraceptive use, and alcohol use). Results are expressed as the ratio of LINE-1 methylation relative to that of the referent group, with the corresponding 95% confidence intervals (CI). We found no significant associations between plasma or breast folate and percent LINE-1 methylation. Variation in MTHFR, MTR, and MTRR were significantly associated with percent LINE-1 methylation. Variant allele carriers of MTHFR A1289C had 4% lower LINE-1 methylation (Ratio 0.96, 95% CI 0.93–0.98), while variant allele carriers of MTR A2756G (Ratio 1.03, 95% CI 1.01–1.06) and MTRR A66G (Ratio 1.03, 95% CI 1.01–1.06) had 3% higher LINE-1 methylation, compared to those carrying the more common genotypes of these SNPs. DNA methylation of LINE-1 elements in histologically normal breast tissues is influenced by polymorphisms in genes in the one-carbon metabolism pathway. Future studies are needed to investigate the sociodemographic, environmental and additional genetic determinants of DNA methylation in breast tissues and the impact on breast cancer susceptibility.

Discovery and replication of microRNAs for breast cancer risk using genome-wide profiling

Background Genome-wide miRNA expression may be useful for predicting breast cancer risk and/or for the early detection of breast cancer. Results A 41-miRNA model distinguished breast cancer risk in the discovery study (accuracy of 83.3%), which was replicated in the independent study (accuracy = 63.4%, P=0.09). Among the 41 miRNA, 20 miRNAs were detectable in serum, and predicted breast cancer occurrence within 18 months of blood draw (accuracy 53%, P=0.06). These risk-related miRNAs were enriched for HER-2 and estrogen-dependent breast cancer signaling. Materials and Methods MiRNAs were assessed in two cross-sectional studies of women without breast cancer and a nested case-control study of breast cancer. Using breast tissues, a multivariate analysis was used to model women with high and low breast cancer risk (based upon Gail risk model) in a discovery study of women without breast cancer (n=90), and applied to an independent replication study (n=71). The model was then assessed using serum samples from the nested case-control study (n=410). Conclusions Studying breast tissues of women without breast cancer revealed miRNAs correlated with breast cancer risk, which were then found to be altered in the serum of women who later developed breast cancer. These results serve as proof-of-principle that miRNAs in women without breast cancer may be useful for predicting breast cancer risk and/or as an adjunct for breast cancer early detection. The miRNAs identified herein may be involved in breast carcinogenic pathways because they were first identified in the breast tissues of healthy women.

Genetic Variation in Adipokine Genes and Associations with Adiponectin and Leptin Concentrations in Plasma and Breast Tissue

Background: Circulating adipokines may be associated with breast cancer risk. Genetic variants governing adipokines and adipokine receptors may also predict risk, but their effect on breast adipokine concentrations is unknown. Methods: We conducted a cross-sectional analysis of functional SNPs in 5 adipokine genes [adiponectin, leptin (LEP), and their receptors] among 85 cancer-free women who were undergoing reduction mammoplasty. Results: In multivariable-adjusted regression models, compared with the common GG genotype, the AA genotype of the LEP A19G SNP was associated with 27% lower plasma adiponectin [ratio, 0.73; 95% confidence interval (CI), 0.54–0.98] and leptin (ratio, 0.73; 95% CI, 0.55–0.96). Women with the AG genotype of LEP A19G had 39% lower breast leptin (ratio, 0.61; 95% CI, 0.39–0.97) compared with those with the GG genotype. No associations were observed for SNPs in the remaining genes. Conclusions: Genetic variation in LEP may alter endogenous adipokine concentrations in circulation and in breast tissues. Impact: These preliminary findings may support the hypothesis that genetic variation in adipokine genes modifies circulating adipokine concentrations and possibly leptin concentrations in local breast tissues, which may be associated with breast cancer risk. Cancer Epidemiol Biomarkers Prev; 23(8); 1559–68. ©2014 AACR.

MicroRNAs Role in Prostate Cancer

Prostate cancer still represents a major health problem for men worldwide. Due to the specific limitation of the currently used clinical biomarkers for prostate cancer, there is a need to identify new and more accurate prostate-specific biomarkers, both for diagnosis and prediction. Small noncoding species of RNAs called microRNAs (miRNAs) have emerged as possible biomarkers in cancer tissues as well as biological fluids, including for prostate cancer. Moreover, it has been shown that miRNAs could be used as therapeutic targets in different cancer types, including prostate cancer, playing an important role in improving diagnosis and prognosis; and miRNAs have the potential to be clinically useful as predictors of response to personalized cancer therapy and as predictors of prognosis. The analysis of miRNAs in prostate tissue is rather straightforward and has been routinely done on fresh tissue. In addition, due to the more stable nature of miRNAs, they are amenable to be analyzed in archived formalin fixed paraffin embedded tissue as well, and also in serum, plasma and urine, using various analytical platforms including microarrays, next generation sequencing and real time PCR. Moreover, although the existence or prostasomes (microvesicles secreted by prostate cells including prostate cancer cells) has been known for years and they were studied as a source of biomarkers for prostate cancer, only recently it has been described that these vesicles also contain miRNAs that could be used as biomarkers in prostate cancer. This chapter underscores the feasibility of current technologies for miRNA analysis and their importance in prostate cancer biology. Moreover, elucidating the specific alteration of miRNA expression and how to modulate it in prostate tissue will open new avenues for developing therapeutic strategies for prostate cancer treatment.

Abstract 2777: One-carbon metabolism genetic variant and genome-wide DNA methylation in breast tissues from healthy women

Altered DNA methylation is an early event in carcinogenesis. Little is known about the mechanism of altered methylation in breast tissue; possible factors include diet such as alcohol and folate intake, and genetic variation for enzymes in one carbon metabolism. Examination of the association of these factors with methylation in breast tissues from healthy women provides insight into these changes. Blood and glandular breast tissues from 81 women with no history of cancer and who underwent reduction mammoplasty were assayed. The 96-plex Illumina BeadXpress® or TaqMan® SNP Genotyping assays assessed SNPs, genome-wide DNA methylation profiling was performed using the Illumina Infinium HumanMethylation450 BeadChip.The Affymetrix GeneChip Human Trascriptome Array 2.0 was used to compare gene expression level with methylation change in fresh frozen breast tissues. Biological networks of differentially-methylated (DM) genes were assigned using the Ingenuity Pathway Analysis (IPA). Fifty-seven CpG sites were DM in comparisons of genotype for eight SNPs in FTHFD, MTHFD1, MTHFR, MTR, MTRR, and TYMS (P Citation Format: Min-Ae Song, Theodore M. Brasky, Catalin Marian, Daniel Y. Weng, Cenny Taslim, Adana A. Llanos, Ramona G. Dumitrescu, Zhenhua Liu, Joel B. Mason, Bhaskar V. Kallakury, Jo L. Freudenheim, Peter G. Shields. One-carbon metabolism genetic variant and genome-wide DNA methylation in breast tissues from healthy women. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2777.