Effects of Pubertal Exposure to Butyl Benzyl Phthalate, Perfluorooctanoic Acid, and Zeranol on Mammary Gland Development and Tumorigenesis in Rats.

Endocrine-disrupting chemicals (EDCs)-including butyl benzyl phthalate (BBP), perfluorooctanoic acid (PFOA), and zeranol (α-ZAL, referred to as ZAL hereafter)-can interfere with the endocrine system and produce adverse effects. It remains unclear whether pubertal exposure to low doses of BBP, PFOA, and ZAL has an impact on breast development and tumorigenesis. We exposed female Sprague Dawley rats to BBP, PFOA, or ZAL through gavage for 21 days, starting on day 21, and analyzed their endocrine organs, serum hormones, mammary glands, and transcriptomic profiles of the mammary glands at days 50 and 100. We also conducted a tumorigenesis study for rats treated with PFOA and ZAL using a 7,12-dimethylbenz[a]anthracene (DMBA) model. Our results demonstrated that pubertal exposure to BBP, PFOA, and ZAL affected endocrine organs and serum hormones, and induced phenotypic and transcriptomic changes. The exposure to PFOA + ZAL induced the most phenotypic and transcriptomic changes in the mammary gland. PFOA + ZAL downregulated the expression of genes related to development at day 50, whereas it upregulated genes associated with tumorigenesis at day 100. PFOA + ZAL exposure also decreased rat mammary tumor latency, reduced the overall survival of rats after DMBA challenge, and affected the histopathology of mammary tumors. Therefore, our study suggests that exposure to low doses of EDCs during the pubertal period could induce changes in the endocrine system and mammary gland development in rats. The inhibition of mammary gland development by PFOA + ZAL might increase the risk of developing mammary tumors through activation of signaling pathways associated with tumorigenesis.

Genomic signature of parity in the breast of premenopausal women.

BACKGROUND: Full-term pregnancy (FTP) at an early age confers long-term protection against breast cancer. Previously, we reported that a FTP imprints a specific gene expression profile in the breast of postmenopausal women. Herein, we evaluated gene expression changes induced by parity in the breast of premenopausal women. METHODS: Gene expression profiling of normal breast tissue from 30 nulliparous (NP) and 79 parous (P) premenopausal volunteers was performed using Affymetrix microarrays. In addition to a discovery/validation analysis, we conducted an analysis of gene expression differences in P vs. NP women as a function of time since last FTP. Finally, a laser capture microdissection substudy was performed to compare the gene expression profile in the whole breast biopsy with that in the epithelial and stromal tissues. RESULTS: Discovery/validation analysis identified 43 differentially expressed genes in P vs. NP breast. Analysis of expression as a function of time since FTP revealed 286 differentially expressed genes (238 up- and 48 downregulated) comparing all P vs. all NP, and/or P women whose last FTP was less than 5 years before biopsy vs. all NP women. The upregulated genes showed three expression patterns: (1) transient: genes upregulated after FTP but whose expression levels returned to NP levels. These genes were mainly related to immune response, specifically activation of T cells. (2) Long-term changing: genes upregulated following FTP, whose expression levels decreased with increasing time since FTP but did not return to NP levels. These were related to immune response and development. (3) Long-term constant: genes that remained upregulated in parous compared to nulliparous breast, independently of time since FTP. These were mainly involved in development/cell differentiation processes, and also chromatin remodeling. Lastly, we found that the gene expression in whole tissue was a weighted average of the expression in epithelial and stromal tissues. CONCLUSIONS: Genes transiently activated by FTP may have a role in protecting the mammary gland against neoplastically transformed cells through activation of T cells. Furthermore, chromatin remodeling and cell differentiation, represented by the genes that are maintained upregulated long after the FTP, may be responsible for the lasting preventive effect against breast cancer.

BC200 overexpression contributes to luminal and triple negative breast cancer pathogenesis.

BACKGROUND: Long non coding RNAs (lncRNAs) are RNA molecules longer than 200 nucleotides that are not translated into proteins, but regulate the transcription of genes involved in different cellular processes, including cancer. Epidemiological analyses have demonstrated that parous women have a decreased risk of developing breast cancer in postmenopausal years if they went through a full term pregnancy in their early twenties. We here provide evidence of the role of BC200 in breast cancer and, potentially, in pregnancy's preventive effect in reducing the lifetime risk of developing breast cancer. METHODS: Transcriptome analysis of normal breast of parous and nulliparous postmenopausal women revealed that several lncRNAs are differentially expressed in the parous breast. RNA sequencing of healthy postmenopausal breast tissue biopsies from eight parous and eight nulliparous women showed that there are 42 novel lncRNAs differentially expressed between these two groups. Screening of several of these 42 lncRNAs by RT-qPCR in different breast cancer cell lines, provided evidence that one in particular, lncEPCAM (more commonly known as BC200), was a strong candidate involved in cancer progression. Proliferation, migration, invasion and xerograph studies confirmed this hypothesis. RESULTS: The poorly studied oncogenic BC200 was selected to be tested in vitro and in vivo to determine its relevance in breast cancer and also to provide us with an understanding of its role in the increased susceptibility of the nulliparous women to cancer. Our results show that BC200 is upregulated in nulliparous women, and breast cancer cells and tissue. The role of BC200 is not completely understood in any of the breast cancer subtypes. We here provide evidence that BC200 has a role in luminal breast cancer as well as in the triple negative breast cancer subtype. CONCLUSION: When overexpressed in luminal and triple negative breast cancer cell lines, BC200 shows increased proliferation, migration, and invasion in vitro. In vivo, overexpression of BC200 increased tumor size. Although treatment for cancer using lncRNAs as targets is in its infancy, the advancement in knowledge and technology to study their relevance in disease could lead to the development of novel treatment and preventive strategies for breast cancer.

Aspirin abrogates impairment of mammary gland differentiation induced by early in life second-hand smoke in mice.

Epidemiological studies show that there is limited evidence that tobacco smoking causes breast cancer in humans. In rodents, many tobacco smoke chemicals cause mammary gland tumors. This study evaluated the mammary gland differentiation in mice exposed to environmental cigarette smoke (ECS), using 3R4F Kentucky reference cigarettes, starting after birth and continuing daily for 10 weeks (total particulate exposure 95 mg/m3; CO 610 ppm). We also analyzed the effects of oral administration of non-steroidal anti-inflammatory drugs (NSAIDs), aspirin (1600 mg/kg) or naproxen (320 mg/kg), on mammary gland differentiation, either in unexposed or ECS-exposed mice. The ECS exposure caused delay of mammary glands development. We speculate that this delay may result from aryl hydrocarbon receptor (AHR) signaling activation, which has an antiestrogenic effect and crosstalk to the estrogen metabolism pathway. Similarly, naproxen impaired gland differentiation in unexposed and ECS-exposed mice, while aspirin hindered its development only in unexposed mice. The lack of differentiation induced by the NSAIDs could be explained by their antiestrogenic effect through inhibition of aldo-keto reductases. In ECS-exposed animals, aspirin induced intense lobular formation, which could indicate that aspirin is counteracting the AHR signaling induced by ECS. Based on the differentiation induced by aspirin in ECS-exposed animals, we postulate that these mice would be less susceptible to mammary carcinogenesis. Our results suggest that exposure to smoke at an early age impairs the development of the mammary gland, thus resulting in a longer period of susceptibility and increased risk of breast cancer. However, addition of aspirin can abrogate this effect.

Recombinant human chorionic gonadotropin induces signaling pathways towards cancer prevention in the breast of BRCA1/2 mutation carriers.

BACKGROUND: Strategies for breast cancer prevention in women with germline BRCA1/2 mutations are limited. We previously showed that recombinant human chorionic gonadotropin (r-hCG) induces mammary gland differentiation and inhibits mammary tumorigenesis in rats. The present study investigated hCG-induced signaling pathways in the breast of young nulliparous women carrying germline BRCA1/2 mutations. METHODS: We performed RNA-sequencing on breast tissues from 25 BRCA1/2 mutation carriers who received r-hCG treatment for 3 months in a phase II clinical trial, we analyzed the biological processes, reactome pathways, canonical pathways, and upstream regulators associated with genes differentially expressed after r-hCG treatment, and validated genes of interest. RESULTS: We observed that r-hCG induces remarkable transcriptomic changes in the breast of BRCA1/2 carriers, especially in genes related to cell development, cell differentiation, cell cycle, apoptosis, DNA repair, chromatin remodeling, and G protein-coupled receptor signaling. We revealed that r-hCG inhibits Wnt/ß-catenin signaling, MYC, HMGA1 , and HOTAIR , whereas activates TGFB/TGFBR-SMAD2/3/4, BRCA1, TP53, and upregulates BRCA1 protein. CONCLUSION: Our data suggest that the use of r-hCG at young age may reduce the risk of breast cancer in BRCA1/2 carriers by inhibiting pathways associated with stem/progenitor cell maintenance and neoplastic transformation, whereas activating genes crucial for breast epithelial differentiation and lineage commitment, and DNA repair.

Pregnancy-induced chromatin remodeling in the breast of postmenopausal women.

Early pregnancy and multiparity are known to reduce the risk of women to develop breast cancer at menopause. Based on the knowledge that the differentiation of the breast induced by the hormones of pregnancy plays a major role in this protection, this work was performed with the purpose of identifying what differentiation-associated molecular changes persist in the breast until menopause. Core needle biopsies (CNB) obtained from the breast of 42 nulliparous (NP) and 71 parous (P) postmenopausal women were analyzed in morphology, immunocytochemistry and gene expression. Whereas in the NP breast, nuclei of epithelial cells were large and euchromatic, in the P breast they were small and hyperchromatic, showing strong methylation of histone 3 at lysine 9 and 27. Transcriptomic analysis performed using Affymetrix HG_U133 oligonucleotide arrays revealed that in CNB of the P breast, there were 267 upregulated probesets that comprised genes controlling chromatin organization, transcription regulation, splicing machinery, mRNA processing and noncoding elements including XIST. We concluded that the differentiation process induced by pregnancy is centered in chromatin remodeling and in the mRNA processing reactome, both of which emerge as important regulatory pathways. These are indicative of a safeguard step that maintains the fidelity of the transcription process, becoming the ultimate mechanism mediating the protection of the breast conferred by full-term pregnancy.

Fish oil alters tamoxifen-modulated expression of mRNAs that encode genes related to differentiation, proliferation, metastasis, and immune response in rat mammary tumors.

We have previously shown that a fish oil (FO)-rich diet increased the chemopreventive efficacy of tamoxifen (Tam) against N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinogenesis. Herein, we provide evidence that Tam treatment modifies gene expression of mammary tumors depending upon the type of dietary fat fed to the animals. Rats initiated with MNU and treated with Tam were fed a diet rich in corn oil or FO. After 8 wk, cribriform tumors were collected and gene expression analysis was performed. Increased RNA expression of genes such as SerpinB10, Wisp2, and Apod in tumors from FO-treated rats is indicative of highly differentiated tumors. Decreased expression of H19 and Igf2 mRNA in Tam-treated groups, and Gamma Synuclein mRNA in the FO + Tam group may be related to tumor growth impairment and lower metastatic capacity. Change in the expression of genes associated with immunity in animals in the FO + Tam group may suggest a shift in the immune response. These data show that, although Tam modulates the expression of genes leading to tumor growth impairment, further modulations of genes are influenced by FO. FO modulation of Tam changes in gene expression accounts for its enhancing chemopreventive effect against MNU-induced mammary carcinogenesis. Supplemental materials are available for this article. Go to the publisher's online edition of Nutrition and Cancer to view the supplemental file.

An in vitro-in vivo model of epithelial mesenchymal transition in triple negative breast cancer.

The loss of epithelial expression markers by neoplastic breast cancer cells in the primary tumor is believed to play a pivotal role during breast cancer metastasis. This phenomenon is the hallmark of the epithelial mesenchymal transition (EMT) process. Gene expression microarrays were performed to investigate key functional elements on an in vitro metastasis model derived from human breast epithelial cells (MCF10F) treated with 17 beta estradiol. We identified groups of SLUG associated genes modulated during EMT.

Toward a digital analysis of environmental impacts on rodent mammary gland density during critical developmental windows.

While mammographic breast density is associated with breast cancer risk in humans, there is no comparable surrogate risk measure in mouse and rat mammary glands following various environmental exposures. In the current study, mammary glands from mice and rats subjected to reproductive factors and exposures to environmental chemicals that have been shown to influence mammary gland development and/or susceptibility to mammary tumors were evaluated for histologic density by manual and automated digital methods. Digital histological density detected changes due to hormonal stimuli/reproductive factors (parity), dietary fat, and exposure to environmental chemicals, such as benzophenone-3 and a combination of perfluorooctanoic acid and zeranol. Thus, digital analysis of mammary gland density offers a high throughput method that can provide a highly reproducible means of comparing a measure of histological density across independent experiments, experimental systems, and laboratories. This methodology holds promise for the detection of environmental impacts on mammary gland structure in mice and rats that may be comparable to human breast density, thus potentially allowing comparisons between rodent models and human breast cancer studies.

In utero exposure to butyl benzyl phthalate induces modifications in the morphology and the gene expression profile of the mammary gland: an experimental study in rats.

BACKGROUND: Environmental estrogens are exogenous estrogen-mimicking compounds that can interfere with endogenous endocrine systems. Several of these endocrine disruptors have been shown to alter normal development and influence tumorigenesis in experimental models. N-butyl benzyl phthalate (BBP), a widely used plasticizer, is a well-known endocrine disruptor. The aim of this study was to elucidate the effect of prenatal exposure to BBP on the morphology, proliferative index, and genomic signature of the rat mammary gland at different ages. METHODS: In utero exposure was performed by gavage of pregnant Sprague Dawley CD rats with 120mg or 500mg BBP/kg/day from day 10 post-conception to delivery. Female litters were euthanized at 21, 35, 50 and 100 days. The morphology and proliferative index of the mammary gland were studied from whole mount preparations and BrdU incorporation, respectively. Gene expression profile was assessed by microarrays. Several genes found differentially expressed and related to different functional categories were further validated by real time RT-PCR. RESULTS: Prenatal exposure of BBP induced delayed vaginal opening and changes in the post-natal mammary gland long after the end of the treatment, mainly by 35 days of age. Exposure to the high dose resulted in modifications in architecture and proliferative index of the mammary gland, mostly affecting the undifferentiated terminal end buds. Moreover, the expression profiles of this gland in the exposed rats were modified in a dose-dependent fashion. Analysis of functional categories showed that modified genes were related to immune function, cell signaling, proliferation and differentiation, or metabolism. CONCLUSIONS: Our data suggest that in utero exposure to BBP induced a delayed pubertal onset and modified morphology of the mammary gland. These alterations were accompanied by modifications in gene expression previously associated with an increased susceptibility to carcinogenesis.

Epigenetic reprogramming of epithelial mesenchymal transition in triple negative breast cancer cells with DNA methyltransferase and histone deacetylase inhibitors.

BACKGROUND: Triple negative breast cancer (TNBC) is an aggressive neoplasia with no effective therapy. Our laboratory has developed a unique TNBC cell model presenting epithelial mesenchymal transition (EMT) a process known to be important for tumor progression and metastasis. There is increasing evidence showing that epigenetic mechanisms are involved in the activation of EMT. The objective of this study is to epigenetically reverse the process of EMT in TNBC by using DNA methyltransferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi). METHODS: We evaluated the antitumor effect of three DNMTi and six HDACi using our TNBC cell model by MTT assay, migration and invasion assay, three dimensional culture, and colony formation assay. We then performed the combined treatment both in vitro and in vivo using the most potent DNMTi and HDACi, and tested the combined treatment in a panel of breast cancer cell lines. We investigated changes of EMT markers and potential signaling pathways associated with the antitumor effects. RESULTS: We showed that DNMTi and HDACi can reprogram highly aggressive TNBC cells that have undergone EMT to a less aggressive phenotype. SGI-110 and MS275 are superior to other seven compounds being tested. The combination of SGI with MS275 exerts a greater effect than single agent alone in inhibiting cell proliferation, motility, colony formation, and stemness of cancer cells. We also demonstrated that MS275 and the combination of SGI with MS275 exert in vivo antitumor effect. We revealed that the combined treatment synergistically reverses EMT through inhibiting EpCAM cleavage and WNT signaling, suppressing mutant p53, ZEB1, and EZH2, and inducing E-cadherin, apoptosis, as well as histone H3 tri-methylation. CONCLUSIONS: Our study showed that DNMTi and HDACi exert antitumor activity in TNBC cells partially by epigenetically reprograming EMT. Our findings strongly suggest that TNBC is sensitive to epigenetic therapies. Therefore, we propose a new strategy to treat TNBC by using the combination of SGI-110 with MS275, which exerts superior antitumor effects by simultaneously targeting multiple pathways.

DNA Methylation Targets Influenced by Bisphenol A and/or Genistein Are Associated with Survival Outcomes in Breast Cancer Patients.

Early postnatal exposures to Bisphenol A (BPA) and genistein (GEN) have been reported to predispose for and against mammary cancer, respectively, in adult rats. Since the changes in cancer susceptibility occurs in the absence of the original chemical exposure, we have investigated the potential of epigenetics to account for these changes. DNA methylation studies reveal that prepubertal BPA exposure alters signaling pathways that contribute to carcinogenesis. Prepubertal exposure to GEN and BPA + GEN revealed pathways involved in maintenance of cellular function, indicating that the presence of GEN either reduces or counters some of the alterations caused by the carcinogenic properties of BPA. We subsequently evaluated the potential of epigenetic changes in the rat mammary tissues to predict survival in breast cancer patients via the Cancer Genomic Atlas (TCGA). We identified 12 genes that showed strong predictive values for long-term survival in estrogen receptor positive patients. Importantly, two genes associated with improved long term survival, HPSE and RPS9, were identified to be hypomethylated in mammary glands of rats exposed prepuberally to GEN or to GEN + BPA respectively, reinforcing the suggested cancer suppressive properties of GEN.

Combination of Antiestrogens and Omega-3 Fatty Acids for Breast Cancer Prevention.

The molecular and biological heterogeneity of human breast cancer emphasizes the importance of a multitargeted approach for effective chemoprevention. Targeting the estrogen receptor pathway alone with the antiestrogens, Tamoxifen and Raloxifene reduces the incidence of estrogen receptor positive tumors but is ineffective against the development of hormone independent cancers. Our preclinical data indicate that the administration of omega-3 fatty acids potentiates the antitumor effects of Tamoxifen by inhibiting multiple proliferative and antiapoptotic pathways, several of which interact with estrogen receptor signaling. The complementarity in the mechanism of antitumor action of Tamoxifen and omega-3 fatty acids is well supported by our signaling, genomic, and proteomic studies. Furthermore, administration of omega-3 fatty acids allows the use of lower and, hence, likely less toxic doses of Tamoxifen. If these findings are supported in the clinical setting, the combination of omega-3 fatty acids and anteistrogens may emerge as a promising, effective, and safe chemopreventive strategy to be tested in a large multi-institutional trial using breast cancer incidence as the primary endpoint.

Molecular pathways involved in pregnancy-induced prevention against breast cancer.

Pregnancy produces a protective effect against breast cancer in women who had their first full term pregnancy (FTP) in their middle twenties. The later in life the first delivery occurs, the higher the risk of breast cancer development. Also, transiently during the postpartum period, the risk of developing breast cancer increases. This transient increased risk is taken over by a long-lasting protective period. The genomic profile of parous women has shown pregnancy induces a long-lasting "genomic signature" that explains the preventive effect on breast cancer. This signature reveals that chromatin remodeling is the driver of the differentiation process conferred by FTP. The chromatin remodeling process may be the ultimate step mediating the protection of the breast against developing breast cancer in post-menopausal years.

The genomic signature of breast cancer prevention.

The breast of parous postmenopausal women exhibits a specific signature that has been induced by a full term pregnancy. This signature is centered in chromatin remodeling and the epigenetic changes induced by methylation of specific genes which are important regulatory pathways induced by pregnancy. Through the analysis of the genes found to be differentially methylated between women of varying parity, multiple positions at which beta-catenin production and use is inhibited were recognized. The biological importance of the pathways identified in this specific population cannot be sufficiently emphasized because they could represent a safeguard mechanism mediating the protection of the breast conferred by full term pregnancy.

Mimicking pregnancy as a strategy for breast cancer prevention.

Pregnancy and its effects on breast cancer risk have been widely investigated; there is consensus among researchers that early pregnancy confers protection against breast cancer later in life, whereas nulliparity and late-age parity have been associated with increased risk of developing breast cancer. The answer to the question of how pregnancy reduces breast cancer risk has been elusive; however, pregnancy, like breast cancer, is a similar hormone-dependent entity under direct control of estrogen, progesterone and, of particular importance, human chorionic gonadotropin (hCG). In this report, we emphasize the main changes, previously described by our laboratory, in morphology and gene expression levels of the mammary gland of Sprague-Dawley rats exposed to known cancer-preventative conditions (pregnancy, hCG and progesterone + estrogen). In addition, we postulate a protective mechanism induced by hCG that could reduce the cell's potential to be transformed by carcinogens.

Defining the genomic signature of the parous breast.

BACKGROUND: It is accepted that a woman's lifetime risk of developing breast cancer after menopause is reduced by early full term pregnancy and multiparity. This phenomenon is thought to be associated with the development and differentiation of the breast during pregnancy. METHODS: In order to understand the underlying molecular mechanisms of pregnancy induced breast cancer protection, we profiled and compared the transcriptomes of normal breast tissue biopsies from 71 parous (P) and 42 nulliparous (NP) healthy postmenopausal women using Affymetrix Human Genome U133 Plus 2.0 arrays. To validate the results, we performed real time PCR and immunohistochemistry. RESULTS: We identified 305 differentially expressed probesets (208 distinct genes). Of these, 267 probesets were up- and 38 down-regulated in parous breast samples; bioinformatics analysis using gene ontology enrichment revealed that up-regulated genes in the parous breast represented biological processes involving differentiation and development, anchoring of epithelial cells to the basement membrane, hemidesmosome and cell-substrate junction assembly, mRNA and RNA metabolic processes and RNA splicing machinery. The down-regulated genes represented biological processes that comprised cell proliferation, regulation of IGF-like growth factor receptor signaling, somatic stem cell maintenance, muscle cell differentiation and apoptosis. CONCLUSIONS: This study suggests that the differentiation of the breast imprints a genomic signature that is centered in the mRNA processing reactome. These findings indicate that pregnancy may induce a safeguard mechanism at post-transcriptional level that maintains the fidelity of the transcriptional process.

Human chorionic gonadotropin and a 15 amino acid hCG fragment of the hormone induce downregulation of the cytokine IL-8 receptor in normal breast epithelial cells.

Cytokine receptors are associated with tumor cell growth by increasing proliferation, metastasis and regulating self-renewal of cancer stem cells (SCs). There is a strong association between cytokine IL-8 receptor (CXCR1) over-expression and cells displaying SC characteristics. Human chorionic gonadotropin (hCG) causes differentiation, inhibition of cell proliferation and increased apoptosis of the breast epithelium. hCG receptor (LHCGR) expression in breast tumors and in breast cancer cell lines is undetectable or low. In this study, our objective was to assess and compare the effects of hCG and a 15 amino acid hCG fragment of the hormone on mRNA expression of CXCR1 and LHCGR on normal breast epithelial cells (MCF-10F) by real time RT-PCR after treatment with hCG or a hCG fragment for 15 days. Cell proliferation was also measured. hCG and the hCG fragment decreased cell proliferation in both groups. The compounds upregulated LHCGR expression and downregulated CXCR1 expression. It is possible to postulate that an increase of LHCGR mRNA seems to respond to the decrease of CXCR1 expression. These genes probably act synergistically to reduce the amount of cancer SCs in the mammary gland. Thereby, the use of hCG or the hCG fragment as a therapeutic or preventive tool should be considered.

Characterization of a genomic signature of pregnancy identified in the breast.

The objective of this study was to comprehensively compare the genomic profiles in the breast of parous and nulliparous postmenopausal women to identify genes that permanently change their expression following pregnancy. The study was designed as a two-phase approach. In the discovery phase, we compared breast genomic profiles of 37 parous with 18 nulliparous postmenopausal women. In the validation phase, confirmation of the genomic patterns observed in the discovery phase was sought in an independent set of 30 parous and 22 nulliparous postmenopausal women. RNA was hybridized to Affymetrix HG_U133 Plus 2.0 oligonucleotide arrays containing probes to 54,675 transcripts, scanned and the images analyzed using Affymetrix GCOS software. Surrogate variable analysis, logistic regression, and significance analysis of microarrays were used to identify statistically significant differences in expression of genes. The false discovery rate (FDR) approach was used to control for multiple comparisons. We found that 208 genes (305 probe sets) were differentially expressed between parous and nulliparous women in both discovery and validation phases of the study at an FDR of 10% and with at least a 1.25-fold change. These genes are involved in regulation of transcription, centrosome organization, RNA splicing, cell-cycle control, adhesion, and differentiation. The results provide initial evidence that full-term pregnancy induces long-term genomic changes in the breast. The genomic signature of pregnancy could be used as an intermediate marker to assess potential chemopreventive interventions with hormones mimicking the effects of pregnancy for prevention of breast cancer.