Age-associated gene expression in normal breast tissue mirrors qualitative age-at-incidence patterns for breast cancer.

BACKGROUND: Age is the strongest breast cancer risk factor, with overall breast cancer risk increasing steadily beginning at approximately 30 years of age. However, while breast cancer risk is lower among younger women, young women's breast cancer may be more aggressive. Although, several genomic and epidemiologic studies have shown higher prevalence of aggressive, estrogen-receptor negative breast cancer in younger women, the age-related gene expression that predisposes to these tumors is poorly understood. Characterizing age-related patterns of gene expression in normal breast tissues may provide insights on etiology of distinct breast cancer subtypes that arise from these tissues. METHODS: To identify age-related changes in normal breast tissue, 96 tissue specimens from patients with reduction mammoplasty, ages 14 to 70 years, were assayed by gene expression microarray. RESULTS: Significant associations between gene expression levels and age were identified for 802 probes (481 increased, 321 decreased with increasing age). Enriched functions included "aging of cells," "shape change," and "chemotaxis," and enriched pathways included Wnt/beta-catenin signaling, Ephrin receptor signaling, and JAK/Stat signaling. Applying the age-associated genes to publicly available tumor datasets, the age-associated pathways defined two groups of tumors with distinct survival. CONCLUSION: The hazard rates of young-like tumors mirrored that of high-grade tumors in the Surveillance, Epidemiology, and End Results Program, providing a biologic link between normal aging and age-related tumor aggressiveness. IMPACT: These data show that studies of normal tissue gene expression can yield important insights about the pathways and biologic pressures that are relevant during tumor etiology and progression.

Paralemmin-1 is over-expressed in estrogen-receptor positive breast cancers.

BACKGROUND: Paralemmin-1 is a phosphoprotein lipid-anchored to the cytoplasmic face of membranes where it functions in membrane dynamics, maintenance of cell shape, and process formation. Expression of paralemmin-1 and its major splice variant (Δ exon 8) as well as the extent of posttranslational modifications are tissue- and development-specific. Paralemmin-1 expression in normal breast and breast cancer tissue has not been described previously. RESULTS: Paralemmin-1 mRNA and protein expression was evaluated in ten breast cell lines, 26 primary tumors, and 10 reduction mammoplasty (RM) tissues using real time RT-PCR. Paralemmin-1 splice variants were assessed in tumor and RM tissues using a series of primers and RT-PCR. Paralemmin-1 protein expression was examined in cell lines using Western Blots and in 31 ductal carcinomas in situ, 65 infiltrating ductal carcinomas, and 40 RM tissues using immunohistochemistry. Paralemmin-1 mRNA levels were higher in breast cancers than in RM tissue and estrogen receptor (ER)-positive tumors had higher transcript levels than ER-negative tumors. The Δ exon 8 splice variant was detected more frequently in tumor than in RM tissues. Protein expression was consistent with mRNA results showing higher paralemmin-1 expression in ER-positive tumors. CONCLUSIONS: The differential expression of paralemmin-1 in a subset of breast cancers suggests the existence of variation in membrane dynamics that may be exploited to improve diagnosis or provide a therapeutic target.

Normal breast tissue of obese women is enriched for macrophage markers and macrophage-associated gene expression.

Activation of inflammatory pathways is one plausible mechanism underlying the association between obesity and increased breast cancer risk. However, macrophage infiltration and local biomarkers of inflammation in breast adipose tissue have seldom been studied in association with obesity. Gene expression profiles of normal breast tissue from reduction mammoplasty patients were evaluated by whole genome microarrays to identify patterns associated with obesity status (normal-weight, body mass index (BMI) <25; overweight, BMI 25-29.9; obese, BMI ≥30). The presence of macrophage-enriched inflammatory loci with immunopositivity for CD68 protein was evaluated by immunohistochemistry (IHC). After adjusting for confounding by age, 760 genes were differentially expressed (203 up and 557 down; FDR = 0.026) between normal-weight and obese women. Gene ontology analysis suggested significant enrichment for pathways involving IL-6, IL-8, CCR5 signaling in macrophages and RXRα and PPARα activation, consistent with a pro-inflammatory state and suggestive of macrophage infiltration. Gene set enrichment analysis also demonstrated that the genomic signatures of monocytes and macrophages were over-represented in the obese group with FDR of 0.08 and 0.13, respectively. Increased macrophage infiltration was confirmed by IHC, which showed that the breast adipose tissue of obese women had higher average macrophage counts (mean = 8.96 vs. 3.56 in normal-weight women) and inflammatory foci counts (mean = 4.91 vs. 2.67 in normal-weight women). Obesity is associated with local inflammation and macrophage infiltration in normal human breast adipose tissues. Given the role of macrophages in carcinogenesis, these findings have important implications for breast cancer etiology and progression.

Quantitative analysis of promoter methylation in exfoliated epithelial cells isolated from breast milk of healthy women.

Promoter methylation analysis of genes frequently silenced in breast cancer is a promising indicator of breast cancer risk, as these methylation events are thought to occur long before presentation of disease. The numerous exfoliated epithelial cells present in breast milk may provide the breast epithelial DNA needed for detailed methylation analysis and assessment of breast cancer risk. Fresh breast milk samples and health, lifestyle, and reproductive history questionnaires were collected from 111 women. Pyrosequencing analysis was conducted on DNA isolated from the exfoliated epithelial cells immunomagnetically separated from the total cell population in the breast milk of 102 women. A total of 65 CpG sites were examined in six tumor suppressor genes: PYCARD (also known as ASC or TMS1), CDH1, GSTP1, RBP1 (also known as CRBP1), SFRP1, and RASSF1. A sufficient quantity of DNA was obtained for meaningful analysis of promoter methylation; women donated an average of 86 ml of milk with a mean yield of 32,700 epithelial cells per ml. Methylation scores were in general low as expected of benign tissue, but analysis of outlier methylation scores revealed a significant relationship between breast cancer risk, as indicated by previous biopsy, and methylation score for several CpG sites in CDH1, GSTP1, SFRP1, and RBP1. Methylation of RASSF1 was positively correlated with women's age irrespective of her reproductive history. Promoter methylation patterns in DNA from breast milk epithelial cells can likely be used to assess breast cancer risk. Additional studies of women at high breast cancer risk are warranted.

Sensitivity to DNA damage is a common component of hormone-based strategies for protection of the mammary gland.

An early full-term pregnancy significantly reduces the risk of getting breast cancer in women. In animals, this protection can be mimicked by a short-term exposure to physiologic doses of estrogen plus progesterone. Sensitization of p53 and up-regulation of transforming growth factor beta are believed to be important aspects of the mechanism by which protection is imparted. Little is known, however, about the use of this pathway in response to other chemopreventive agents. In this article, we investigated the ability of retinoids, such as 9-cis retinoic acid, all-trans retinoic acid, and N-4-hydroxyphenylretinamide (4-HPR), to sensitize the ductal epithelial cells of virgin mammary glands to DNA damage responses. Using a whole-organ culture system, we observed enhanced cell death in response to gamma-irradiation in the virgin tissues treated with retinoids for 72 hours. These retinoids were partially dependent on p53 and transforming growth factor beta to exert their radiosensitizing effects. However, 4-HPR seemed to sensitize other cells or activate these pathways in a different manner as costimulation with ovarian hormones and 4-HPR was additive, whereas coculture of ovarian hormones and the natural retinoids did not increase amount of death. Taken together, these data suggest that sensitization of the mammary epithelium to p53-dependent apoptosis is a common pathway, which is engaged by retinoids as well as ovarian hormones.

Activation of p53, inhibition of telomerase activity and induction of estrogen receptor beta are associated with the anti-growth effects of combination of ovarian hormones and retinoids in immortalized human mammary epithelial cells.

BACKGROUND: A full-term pregnancy has been associated with reduced risk for developing breast cancer. In rodent models, the protective effect of pregnancy can be mimicked with a defined regimen of estrogen and progesterone combination (E/P). However, the effects of pregnancy levels of E/P in humans and their underlying mechanisms are not fully understood. In this report, we investigated the growth inhibitory effects of pregnancy levels of E/P and both natural and synthetic retinoids in an immortalized human mammary epithelial cell line, 76N TERT cell line. RESULTS: We observed that cell growth was modestly inhibited by E/P, 9-cis-retinoic acid (9-cis RA) or all-trans-retinoic acid (ATRA), and strongly inhibited by N-(4-hydroxyphenyl) retinamide (HPR). The growth inhibitory effects of retinoids were further increased in the presence of E/P, suggesting their effects are additive. In addition, our results showed that both E/P and retinoid treatments resulted in increased RARE and p53 gene activity. We further demonstrated that p53 and p21 protein expression were induced following the E/P and retinoid treatments. Furthermore, we demonstrated that while the telomerase activity was moderately inhibited by E/P, 9-cis RA and ATRA, it was almost completely abolished by HPR treatment. These inhibitions on telomerase activity by retinoids were potentiated by co-treatment with E/P, and correlated well with their observed growth inhibitory effects. Finally, this study provides the first evidence that estrogen receptor beta is up-regulated in response to E/P and retinoid treatments. CONCLUSION: Taken together, our studies show that part of the anti-growth effects of E/P and retinoids is p53 dependent, and involve activation of p53 and subsequent induction of p21 expression. Inhibition of telomerase activity and up-regulation of estrogen receptor beta are also associated with the E/P- and retinoid-mediated growth inhibition. Our studies also demonstrate that the potency of retinoids on cell growth inhibition may be increased through combination of estrogen and progesterone treatment.