Breast cancer risk has traditionally been linked to nulliparity or late first full-term pregnancy, whereas young age at first childbirth, multiparity, and breast-feeding are associated with a reduced risk. Early pregnancy confers protection by inducing breast differentiation, which imprints a specific and permanent genomic signature in experimental rodent models. For testing whether the same phenomenon was detectable in the atrophic breast of postmenopausal parous women, we designed a case-control study for the analysis of the gene expression profile of RNA extracted from epithelial cells microdissected from normal breast tissues obtained from 18 parous and 7 nulliparous women free of breast pathology (controls), and 41 parous and 8 nulliparous women with history of breast cancer (cases). RNA was hybridized to cDNA glass microarrays containing 40,000 genes; arrays were scanned and the images were analyzed using ImaGene software version 4.2. Normalization and statistical analysis were carried out using Linear Models for Microarrays and GeneSight software for hierarchical clustering. The parous control group contained 2,541 gene sequences representing 18 biological processes that were differentially expressed in comparison with the other three groups. Hierarchical clustering of these genes revealed that the combined parity/absence of breast cancer data generated a distinct genomic profile that differed from those of the breast cancer groups, irrespective of parity history, and from the nulliparous cancer-free group, which has been traditionally identified as a high-risk group. The signature that identifies those women in whom parity has been protective will serve as a molecular biomarker of differentiation for evaluating the potential use of preventive agents. (Cancer Epidemiol Biomarkers Prev 2008;17(1):51–66)

More than 300 years elapsed since a striking excess in breast cancer mortality was reported in nuns, in whom the increased risk was attributed to their childlessness (1), until MacMahon et al. (2), in a landmark case-control study, found an almost linear relationship between a woman's risk and the age at which she bore her first child. This work, which included areas of high, intermediate, and low breast cancer risk in seven parts of the world, confirmed that pregnancy had a protective effect that was evident from the early teen years and persisted until the middle twenties (2). Other studies have reported that additional pregnancies and breast-feeding confer greater protection to young women, including a statistically significantly reduced risk of breast cancer in women with deleterious BRCA1 mutations who breast-fed for a cumulative total of >1 year (3, 4). Our studies, designed for unraveling what specific phenomena occurred in the breast during pregnancy for conferring a lifetime protection from developing cancer, led us to the discovery that endogenous endocrinological or environmental influences affecting breast development before the first full-term pregnancy were important modulators of the susceptibility of the breast to undergo neoplastic transformation (5, 6). The fact that exposure of the breast of young nulliparous females to environmental physical agents (7) or chemical toxicants (8, 9) results in a greater rate of cell transformation indicates that the immature breast possesses a greater number of susceptible cells that can become the site of origin of cancer, similarly to what has been reported in experimental animal models (5, 6, 10, 11). In these models, the initiation of cancer is prevented by the differentiation of the mammary gland induced by pregnancy (11, 12). The molecular changes involved in this phenomenon are just starting to be unraveled (13-15). In women, the protection conferred by pregnancy, however, is age specific because a delay in childbearing after age 24 progressively increases the risk of cancer development, which becomes greater than that of nulliparous women when the first full-term pregnancy occurs after 35 years of age (2, 16). The higher breast cancer risk that has been associated with early menarche (17) further emphasizes the importance of the length of the susceptibility “window” that encompasses the period of breast development occurring between menarche and the first pregnancy, when the organ is more susceptible to either undergo complete differentiation under physiologic hormonal stimuli, and hence to be protected from breast cancer, or to suffer genetic or epigenetic damage that might contribute to increasing the lifetime risk of developing breast cancer (9, 18). The damage caused by a single or a combination of putative cancer-causing agents might, in turn, be amplified by the genetic make up of the patient, such as the inheritance of the BRCA1 or BRCA2 susceptibility genes, which influences the pattern of breast development and differentiation and is responsible for at least 5% of all the breast cancer cases (19-21). This postulate is supported by our observations that the architectural pattern of lobular development in parous women with cancer differs from that of parous women without cancer, being similar to that of nulliparous women with or without cancer. Thus, the higher breast cancer risk in parous women might have resulted from either a failure of the breast to fully differentiate under the influence of the hormones of pregnancy (22, 23) and/or stimulation of the growth of foci of transformed cells initiated by early damage or genetic predisposition (9, 18, 20).

Numerous studies have been done for understanding how the dramatic modifications that occur during pregnancy in the pattern of lobular development and differentiation (22, 23), cell proliferation, and steroid hormone receptor content of the breast (24) influence cancer risk. Studies at molecular level using different platforms for global genome analysis have confirmed the universality of this phenomenon in various strains of rats and mice (13-15, 25). For testing the permanence of these changes, we have analyzed the pattern of gene expression occurring during and after pregnancy in rodents. Hierarchical cluster analysis of the genomic profile of rat mammary glands in the 15th and 21st days of pregnancy and at 21 and 42 days postpartum revealed four different patterns of expression in relation to the time of pregnancy (25). During pregnancy, genes related to the secretory properties of the mammary epithelium (Cluster A) become up-regulated, decreasing to control values after 21 and 42 days postpartum. Cluster B includes genes related to the apoptotic pathways, the fatty acid binding protein and cathecol-O-methyltransferase, among others, which become up-regulated from the end of pregnancy until the 21st day postpartum and decreasing thereafter. Cluster C represents differentiation-associated genes whose level of expression continuously and progressively increases with time of pregnancy, reaching their highest levels between 21 and 42 days postpartum, and cluster D comprises genes that are up-regulated around the 15th day of pregnancy and become progressively down-regulated from the end of pregnancy until the 42nd day postpartum (25). These observations confirm at genomic level our previous morphologic and physiologic findings indicating that temporal and sequential changes have to occur in the development of the mammary gland for accomplishing a protective degree of differentiation (11, 12, 25-28). The importance of identifying a specific signature by 42 days postpartum is highlighted by the observations that administration of the polycyclic hydrocarbon 7,12-dimethylbenz(a)anthracene to parous rats results in a markedly reduced tumorigenic response, supporting the concept that the differentiation induced by pregnancy shifts the susceptible “intermediate cells” that originate mammary cancer in the terminal end buds of the virginal gland (5, 10) to transformation-resistant cells (11, 12).

Studies in experimental animal models have been useful for uncovering the sequential genomic changes occurring in the mammary gland in response to the multiple hormonal stimuli of pregnancy that lead to the imprinting of a permanent genomic signature. Work reported here was designed with the purpose of testing whether a similar phenomenon occurs in the atrophic breast of postmenopausal parous women, specifically in the epithelium of lobule type 1 (Lob 1), the site of origin of breast carcinomas (5, 6). Our results support our hypothesis that parous women that had not developed breast cancer after menopause exhibit a genomic “signature” that differs from that present in the breast of parous postmenopausal women with cancer or in nulliparous women who traditionally represent a high breast cancer risk group (1-6).

Patients and Methods for Sample Collection

For this three-center hospital-based study, patients were enrolled from the American Oncologic Hospital of the Fox Chase Cancer Center in Philadelphia, Pennsylvania; Christiana Care Health System, Newark, Delaware; and Somerset Medical Center, Sommerville, New Jersey. The study protocol had been approved by the Institutional Review Board of each participating institution, and written informed consent was obtained from every participant. Patients eligible for the study were postmenopausal women ≥50 years of age and whose menses had naturally ceased 1 year before enrollment. Excluded from this study were women whose ovaries had been surgically removed; who had a history of cancer other than nonmelanoma skin cancer; who were taking medications that could interfere with the study protocol such as estrogens (including Tamoxifen and Raloxifene), progestins, androgens, prednisone, thyroid hormones, and insulin; and women with Alzheimer's disease or severe cognitive deficit and were unable to give informed consent.

Participant Identification. Potential participants were identified by a trained research nurse that carried out daily searches of surgical breast consultation visit summaries at the Breast Evaluation Clinic of the three participating hospitals. Those women that fulfilled the eligibility criteria listed above and who were recommended for a breast biopsy by their treating breast surgeon were selected for the study. Information included in visit summaries, such as age, menopausal status, history of cancer, and current medications, was used to determine if a woman was potentially eligible for this study. A letter was sent to each potential participant describing the study and informing them of their eligibility, which was confirmed in a telephone interview placed within 2 weeks of initial clinical evaluations when biopsies were recommended.

Data and Specimen Collection. Data were collected at preoperative clinic visits before biopsies and during breast biopsy procedures. At the preoperative visits, informed consent was obtained, participants were asked to complete a study questionnaire, and height and weight were measured. Each one of the participating hospitals was provided specifically designed kits for breast tissue collection, which included tissue specimen containers partially filled with 70% ethanol, blood collecting tubes, copies of the eligibility criteria, patient data questionnaires, and labels with coded numbers for the biospecimens and questionnaires. All patients were accessed to a Fox Chase Cancer Center database using the originally assigned coded numbers. Patient names and medical record numbers were known only by the treating physician and authorized personnel at each participating hospital.

Breast tissue specimens were obtained by the operating surgeon following standard procedures for surgical breast biopsies at each site only after tissues were evaluated for presence of tumor, and if present, assessment of tumor size, margin identification, and adequacy of the tissue available for pathologic diagnosis. Normal-appearing tissues were taken from areas at a distance ≥2 cm from any grossly identifiable lesion and immediately fixed in 70% ethanol for 8 h, followed by dehydration, paraffin embedding, sectioning, and staining for histologic analysis and laser capture microdissection following previously described procedures (28). Histopathologic diagnosis of tumor type was made by pathologists at each site (Table 1). Only women diagnosed with invasive breast cancer (cases) or benign breast disease without hyperplasia or atypia (controls) were included in the study. Seventy-four postmenopausal women fulfilled the criteria of eligibility for this study; among them, 59 (80%) were parous and 15 (20%) nulliparous. Eighteen of the 59 parous women that had benign breast biopsies but were free of cancer served as controls and 41 women that had a diagnosis of breast cancer were selected as cases. Among the nulliparous women, 7 were free of cancer (control) and 8 had breast cancer (cases). Average ages at the time of diagnosis and at first birth in the parous cases and controls are shown in Table 2. The number of cases per group represents the distribution of cases at each one of the participating hospitals.

cDNA Human Microarray Analysis

RNA isolation and amplification from laser capture microdissection samples were done as previously described (28). Microarrays were prepared by the Fox Chase Cancer Center National Cancer Institute–supported Microarray Facility. Mirror glass slides were used for robotically spotting 40,000 cDNAs representing 28,000 distinct human transcripts, 10,000 identified by expressed sequence tags, and 2,000 controls and blank spots. Probe construction using direct labeling with random hexamer primer and purification using the QIA-quick PCR purification kit (Qiagen) were done as previously described (28). After the last centrifugation at 13,000 rpm for 1 min, the concentration of the eluted material was determined, then partially dried in a vacuum centrifuge and resuspended in 15 μL of hybridization buffer containing 20× SSC and 0.6 μL of 10% (w/v) SDS. Thereafter, the probes were denatured at 95°C, centrifuged for 3 min at 13,000 rpm, and the products were pipetted onto prehybridized arrays; the slides were coverslipped and placed in hybridization chambers (Gene Machine). Arrays were incubated in a 42°C water bath for 16 to 18 h and subsequently washed with 0.5× SSC, 0.01% (w/v) SDS, followed by 0.06× SSC, at room temperature for 10 min each. The slides were centrifuged for 8 min at 800 rpm (130 × g) at room temperature. The glass microarrays were hybridized, placing in the red channel (labeled with Cy5) the amplified RNA from the breast samples and in the green channel (labeled with Cy3) the human universal reference amplified RNA (Stratagene Technologies, Inc.). Each hybridization compared Cy5-labeled cDNA reverse transcribed from amplified RNA isolated from each patient with the Cy3-labeled cDNA reverse transcribed from a universal human reference amplified RNA sample. Equal amounts of fluorescent probes were used to hybridize the cDNA microarrays in triplicate, and after quality verification in the Nanodrop, replicates from the same sample were combined and redistributed into three separate tubes to have identical replicates. Arrays were read in an Affymetrix 428 fluorescent scanner (MWG) at 10-μm resolution, with variable voltage of the photomultiplier tube for obtaining the maximal signal intensities with <1% (w/v) probe saturation. The resulting images were analyzed using ImaGene software version 4.2 (Biodiscovery).

Data Analysis

Normalization and statistical analysis of the expression data were carried out using Linear Models for Microarray Data (29-31). For detecting the differential expression of genes that might not necessarily be highly expressed, background correction using the “normexp” method in Linear Models for Microarray Data was done for adjusting the local median background estimates, a correction strategy that avoids problems with background estimates that are greater than foreground values and ensures that there were no missing or negative corrected intensities. An offset of 100 was used for both channels to further damp down the variability of log ratios for low-intensity spots. The resulting log ratios were normalized by using the print-tip group Lowess method with span 0.4, as recommended by Smyth (31).

Moderated t statistic was used as the basic statistic for significance analysis; it was computed for each probe and for each contrast (31). False discovery rate was controlled using the BH adjustment of Benjamini and Hochberg (32, 33). All genes with P value below a threshold of 0.05 were selected as differentially expressed, maintaining the proportion of false discoveries in the selected group below the threshold value, in this case 5% (34). Hierarchical clustering was done using GeneSight software (version 2.4; BioDiscovery, Inc.).

Gene Validation by Reverse Transcription-PCR Amplification

Genes that were found to be up-regulated in the parous control breast were validated by real-time reverse transcription-PCR (RT-PCR) using nucleotide sequences that were found using the gene accession number obtained from the cDNA glass microarrays and searching the National Cancer Institute Blast website.1

TaqMan primer and probe sets sequences are listed in Table 4. The sense and antisense primer sequences were designed using Primer3 software2 and synthesized by the DNA Sequencing Facility at the Fox Chase Cancer Center. A β-actin primer was included as a control for gene expression. Primers were labeled with SyBro Green dye (Applied Biosystems); for avoiding competition in the multiplex PCR reaction, tube primer concentrations were limited and standardized. All RT-PCR reactions were done on the ABI Prism 7000 Sequence Detection System using the fluorescent SyBro Green methods (SYBRO Green RT-PCR Master Mix Reagents, all from Applied Biosystems). For each RT-PCR reaction, 100 ng of amplified RNA in a total volume of 50 μL were used. Primer and probe concentrations for target genes were optimized according to the manufacturer's recommended procedure. The following thermal cycling conditions were used: 30 min at 48°C, 10 min at 95°C, and 40 cycles of 15 s; denaturation at 95°C for 60 s; and annealing at 60°C. Each gene was analyzed in triplicate, normalized against β-actin, and expressed in relation to a calibrator sample. Results were expressed as relative gene expression using the ΔCt method, as previously described (28).

Identification of Differentially Expressed Genes in Breast Epithelium

For the analysis of the effect of parity on the genomic profile of epithelial cells from Lob 1, cDNA microarray expression profiling of the 74 breast tissue samples described in Table 1 was done. Genes whose expression changes differed by at least 1.2-fold and that were considered to be statistically significant between nulliparous and parous women with and without cancer using established algorithms were selected for further analysis (33). A total of 2,541 gene sequences were found to be differentially expressed (t test with false discovery rate P < 0.05) in the breast epithelium of the parous control group in comparison with nulliparous control and cases and parous cases. The parous control group had 126 genes up-regulated and 103 down-regulated (Table 3) with respect to the nulliparous control and case groups and to the parous group with breast cancer (cases).

Hierarchical Cluster Analysis

Unsupervised hierarchical clustering done using the expression profiles of 2,541 globally varying genes across the nulliparous and parous data sets representing the four groups revealed that samples clustered primarily based on parity status (Fig. 1). This suggested that the principal source of global variation in gene expression across these data sets was due to genetic differences between women due to reproductive history. This observation suggested that determining which parity-induced gene expression changes were conserved among these highly divergent groups could represent a powerful approach to defining a parity-related gene expression signature. Results of clustering set depicted in Fig. 2A and B indicate that the combined parity and absence of breast cancer data generate a distinct genomic profile that differs from the breast cancer groups, irrespective of parity history, and from the nulliparous cancer-free group, which has been traditionally identified as a high-risk group.

Gene Functional Category Analysis

We measured the relevance of Gene Ontology (GO) terms (35) belonging to the category of biological processes in the breast epithelium of parous women and analyzed the biological significance of those terms that were found to be deregulated in response to an early reproductive event with high statistical significance (Tables 3 and 4). Among the 18 categories identified to contain deregulated genes, the most highly represented biological process was gene transcription, in which 21 (64%) genes were up-regulated and 12 (36%) genes were down-regulated. Higher gene expression was observed in 11 processes that included proteolysis and ubiquitination, cell adhesion, response to exogenous agents, metabolism, DNA repair and replication, RNA processing, apoptosis, miscellaneous processes, antiapoptosis, and chromatin modification, in which the ratios of up-regulated to down-regulated genes ranged from 1.75 to 11 (Table 3). A greater number of genes with lower level of expression were observed in various processes that included: cell transport, protein biosynthesis and metabolism, cell signaling-signal transduction, biological process unknown, and biological process and molecular function unknown. The genes composing these categories are listed in Table 3.

A number of genes that in the arrays of the parous control breast epithelial cells were either significantly up-regulated or not modified by the reproductive process were confirmed by RT-PCR. They included tumor necrosis factor receptor superfamily, member 1A–associated via death domain (TRADD), eukaryotic translation initiation factor 4A, isoform 3 (EIF4A3), suppressor of Ty 5 homologue (S. cerevisiae) (SUPT5H; ref. 35), sex determining region Y (SRY)-box 5 (SOX5), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), homeobox D1 (HOXD1), ephrin B3 (EFNB3), p300/CREB-binding protein (CBP)–associated factor (PCAF), inhibitor of DNA binding 4 (ID4), and Surfeit (Table 4). All genes detected as differentially expressed by the microarray platform were confirmed to be differentially expressed by RT-PCR (P < 0.5), whereas those that did not differ among parous and nulliparous control and cases, such as Surfeit, did not differ in the level of expression by RT-PCR (Table 4).

The present work is the first demonstration that an early first full-term pregnancy imprints in the involuted breast lobules of postmenopausal parous women free of breast cancer a specific genomic signature that significantly differs from that of parous women with cancer and nulliparous women with or without the disease. The cDNA microarray analysis of epithelial RNA of completely involuted lobules, represented by Lob 1, obtained by laser capture microdissection, revealed that these cells express a genomic signature composed of 232 deregulated genes representing 18 functional categories.

The signature is composed of both up-regulated and down-regulated genes. Deregulated genes predominated in the category of transcription, in which 63% were up-regulated and 37% down-regulated. The fact that the number of down-regulated genes was slightly higher in the cell transport, protein biosynthesis metabolism, cell signaling-signal transduction, development and morphogenesis, cell cycle and growth, as well as in those categories in which the biological process and the molecular functions are unknown indicates that down-regulation and/or silencing of gene expression plays an important role in the differentiation of the breast induced by pregnancy, as shown in experimental models (11-15, 25).

Twenty-three genes were found to be significantly up-regulated in the parous breast epithelium in the categories of transcription and chromatin modification, an indication that modifications in transcriptional activity during pregnancy play an important role and become a permanent component of the genomic signature imprinted by this physiologic process in the postmenopausal breast epithelium. More than 2-fold significant increase (P < 0.05) over control values was observed in the bromodomain PHD finger transcription factor (BPTF); SUPT5, which has 50% similarity to yeast SPT5 and is part of a protein complex involved in transcriptional repression by modulating chromatin structure (36); and zinc finger protein 498 (ZNF498), which is involved in the regulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolism. The expression of BPTF has been reported to be lost or significantly reduced in primary carcinomas and in cell lines established from different human carcinomas, supporting our postulate that this gene may play a role in suppression of tumors originating from epithelial tissue (37, 38). ID4, a member of the ID family of proteins (Id1–Id4) that function as dominant-negative regulators of basic helix-loop-helix transcription factors, was increased in the parous women epithelium, as confirmed by RT-PCR that detected significant increase in the levels of expression from 0.21 ± 0.23 in nulliparous controls to 830.28 ± 100.33 in parous controls (Table 4). ID4 mRNA has been reported to be expressed in normal breast epithelium and myoepithelium but to be absent in estrogen receptor α (ER-α)–positive invasive carcinomas, sporadic breast cancers expressing both ER-α and BRCA1 (39), ductal carcinomas in situ, and atypical ductal hyperplasias (40). Epigenetic inactivation of ID4 has been reported in human leukemia (41), colorectal cancer (42), and gastric adenocarcinoma (43). Its complete or partial epigenetic inactivation also occurs in both ER-α–positive and ER-α–negative cells (i.e., T47D, MCF-7, and HBL-100, BT20, BT549, and BR2, respectively; ref. 44), findings that support the role of this gene as a putative tumor-suppressor gene and as a key controller of cell differentiation.

The fact that SRY box 10 or SOX10, a gene that is methylated in the breast cancer cell line MCF7 (45), is significantly up-regulated in the breast of parous women indicates that it may play an integral role in the specification and transcription of the terminal differentiation that has been reported in other systems, such as astrocytes and oligodendrocytes (46). In contrast, SRY box 3 (SOX3), which is involved in the regulation of embryonic development and determination of cell fate (47) and is essential for the maintenance of spermatogonial stem cells (48), is down-regulated in the parous breast. These observations suggest that these genes might play in the breast a role similar to that described in neural and male reproductive organs, respectively. Nevertheless, the final molecular mechanisms by which these transcription factors regulate the differentiation of the parous breast epithelium need further investigation. Transcription factors also associated to coactivators and chromatin remodeling, such PCAF, which have previously found to be significantly up-regulated in breast epithelial cells of parous women (6, 25-27), seem to play an important role in the genomic signature induced by pregnancy in breast epithelial cells. The p300/CBP family of coactivators can interact with the isolated A/B domain of the ER-α, enhancing its AF-1 activity and thus contributing to ligand-independent activity of the receptor under the stimulus of steroid receptor coactivator-1 (49). Interestingly, p300/CBP is recruited by steroid receptor coactivator-1 and cofactors such as transcription intermediary factor 2 and amplified in breast cancer 1, which interact with nuclear receptors in a ligand-dependent manner for enhancing transcriptional activation by the receptor via histone acetylation/methylation (50). PCAF is also a coactivator of the tumor suppressor p53 and participates in p53-mediated transactivation of target genes through acetylation of both bound p53 and histones within p53 target promoters (51). The up-regulation of PCAF in the differentiated breast epithelial cells of parous women might be associated with an increase in the protein levels of the histone acetyl transferases p300, whereas CBP suppresses the level of histone deacetylase (HDAC) and increases the level of acetylated histone H4, as it has been reported for metastatic breast cancer cells after treatment with all-trans retinoic acid, which also up-regulates the expression of BAX (52), a proapoptotic gene that is also up-regulated in the parous breast epithelial cells.

The general transcription factor IIB (GTF2B), which encodes one of the ubiquitous factors required for transcription initiation by RNA polymerase II and HOXD1, is also up-regulated in the parous breast. Of great interest is the fact that transcription factors encoded by the HOX genes, which play a crucial role in Drosophila, Xenopus, and mammalian embryonic differentiation and development, up-regulate HOXC6, HOXD1, and HOXD8 expression in human neuroblastoma cells that are chemically induced to differentiate, an indication that HOX is associated with maturation toward a differentiated neuronal phenotype (53).

Two protein inhibitors of activated STAT (PIAS) were found to be deregulated in the breast epithelium of parous women; PIAS1 was up-regulated and PIAS2 (also called PIASx) was down-regulated. Members of the PIAS protein family have been identified as negative regulators of STAT signaling and of transcription factors such nuclear factor κB and p53 (54). PIAS members have small ubiquitin-like modifier (SUMO) E3-ligase activity, PIAS1 exerting a direct inhibition of STAT1 DNA binding whereas PIAS2 recruiting HDAC3 for repressing STAT4-dependent transcription. Several PIAS can also cause STAT sumoylation, which is likely to inhibit STAT signaling (55). The down-regulation of PIAS2 needs further analysis because the extent of PIAS and SUMO family expression in breast tissues remains unclear, although preliminary evidence suggests that dysregulation of PIAS expression does occur in human breast cancers.

Among the genes that are down-regulated in the involuted lobular epithelium of postmenopausal parous women are HDAC8 and methyl-CpG binding domain protein 3 (MBD3). The importance of the down-regulation of these two genes is highlighted by the fact that HDACs interact with DNA methyltransferases and methyl CpG-binding domain (MBD) proteins, which are associated with CpG island methylation, another epigenetic modification involved in transcriptional repression and heterochromatin remodeling (56-59). The inhibition of HDAC by trichostatin A induces terminal differentiation of mouse erythroleukemia cells and apoptosis of lymphoid and colorectal cancer cells. In addition, trichostatin A treatment of cells expressing the PML zinc finger protein derepresses transcription and allows cells to differentiate normally. These findings have led to the development of HDAC inhibitors as potential agents for the treatment of certain forms of cancer (57). Interestingly, the deacetylase activity of HDAC8 is inhibited by protein kinase A–mediated phosphorylation, resulting in the hyperacetylation of histones H3 and H4, a phenomenon similar to that induced by human chorionic gonadotropin in the human breast (58) and which represents a novel mechanism of regulation of the activities of human class I HDAC by protein kinases (56). MBD3 is one of the five members of the MBD family that recruit various HDAC-containing repressor complexes leading to silencing by generating repressive chromatin structures at relevant binding sites. It plays an important role in mediating the HDAC-specific small-molecule inhibitor (HDI)–induced gene regulations associated with cancer-selective cell death, imparting HDI-induced selectivity in cancer cells via differential transcriptional regulation (59). Silencing of MBD3 abrogates HDI-induced transcriptional reprogramming and growth inhibition in HDI-treated lung cancer cells but not in normal cells. In response to HDI treatment, MBD3 relocalizes within cells in a different manner in cancer and normal cells, an indication that the relocation of MBD3 to the nucleus may facilitate its recruitment to the genome and allow MBD3 to function as a regulatory molecule (59). Our ongoing studies have been designed for clarifying whether intracellular relocation plays a role in differential transcriptional reprogramming in response to pregnancy-induced differentiation.

We found of great interest our findings that genes that are involved in the metabolism of xenobiotic substances and oxidative stress were significantly up-regulated in the breast epithelium of postmenopausal parous women. Among them are epoxide hydrolase or EPHX1, which plays an important role in both the activation and detoxification of exogenous chemicals such as polycyclic aromatic hydrocarbons found in cigarette smoke (60), and thioredoxin reductase 1 or TXNRD1 (61), a member of the pyridine nucleotide family of oxidoreductases and one of the major antioxidant and redox regulators in mammals. TXNRD1 protein reduces thioredoxins and other substrates, playing a role in selenium metabolism, protecting against oxidative stress, and supporting the function of p53 and of other tumor suppressors. The up-regulation in the parous breast epithelium of glutathione S-transferase (GST) 𝛉1 (GSTT1), which belongs to a family of important enzymes involved in the detoxification of a wide variety of known and suspected carcinogens, including potential mammary carcinogens identified in charred meats and tobacco smoke, is of importance because a substantial proportion of the Caucasian population has a homozygous deletion (null) of the GSTM1 or GSTT1 gene, which results in lack of production of these isoenzymes and a significantly elevated risk of breast cancer associated with cigarette smoking (62). N-Acetyltransferase 2-arylamine N-acetyltransferase (NAT2), which is involved in the metabolism of different xenobiotics including potential carcinogens (63), indicates that the lifetime sequel of the differentiation of the breast by an early pregnancy is the activation of a system of defense that makes the parous breast cells less vulnerable to genotoxic substances. This contention is supported by in vitro data showing that breast epithelial cells from parous women do not express phenotypes of cell transformation when treated with chemical carcinogens, whereas those from nulliparous women do (64).

Seven DNA repair controlling genes were found to be significantly up-regulated in the Lob 1 of the parous breast, an indication that an improved DNA repair system was involved in the protective effect induced by pregnancy, as we have previously shown in the rodent experimental model in which mammary epithelial cells of parous animals remove 7,12-dimethylbenz(a)anthracene DNA adducts more efficiently than those of virgin animals (65). DNA repair is central to the integrity of the human genome and reduced DNA repair capacity has been linked to genetic susceptibility to cancer, including that of the breast (66). Among the genes that were up-regulated in the epithelial cells of the parous breast was RAD51-like 3 or RAD51D, which is one of the five RAD51 paralogues that are required in mammalian cells for normal levels of genetic recombination and damaging agents (67). We have previously reported that the X ray repair complementing defective repair I (XRCC4) gene is up-regulated in breast epithelial cells of parous women (6, 26). XRCC4 is a DNA repair factor that is essential for the resolution of DNA double-strand break during V(D)J recombination, acting as a caretaker of the mammalian genome in both normal development and suppression of tumors. In the present study, we found in the same cells the up-regulation of excision repair cross-complementing rodent repair deficiency, complementation group 8 (ERCC8), also known as CSA (68), which interacts with CSB, and when mutated the transcription-coupled repair, a DNA repair defect found in Cockayne syndrome, is impaired (68). Ankyrin repeat domain 17 (ANKRD17), translin or TSN, which encodes a DNA-binding protein that specifically recognizes conserved target sequences at the breakpoint junction of chromosomal translocations (69), and three prime repair exonuclease 1 (TREX1) are also up-regulated in the parous control group. The protein encoded by this latter gene uses two different open reading frames from which the upstream open reading frame encodes proteins that interact with the ataxia telangiectasia and Rad3–related protein, a checkpoint kinase. The proteins encoded by this upstream open reading frame localize to intranuclear foci following DNA damage and are essential components of the DNA damage checkpoint (70, 71). These data indicate that the activation of genes involved in the DNA repair process is part of the signature induced in the mammary gland by pregnancy, confirming previous findings that, in vivo, the ability of the cells to repair carcinogen-induced damage by unscheduled DNA synthesis and adduct removal is more efficient in the post pregnancy mammary gland (65).

Among the genes that control apoptosis, eight were deregulated, six were up-regulated, and two down-regulated. The former included the BCL2-associated X protein (BAX), a proapoptotic gene that belongs to the BCL2 protein family whose transcription is stimulated by the active p53 and the proapoptotic and cell cycle regulator gene p21 (72). To the same category belong the cytotoxic granule–associated RNA binding protein (TIA1), tumor necrosis factor (TNF) receptor–associated factor 1 (TRAF1), TRADD, CASP2 and RIPK1 domain containing adaptor with death domain (CRADD), and protein phosphatase 1F (PPM1F). TIA1 possesses nucleolytic activity against CTL target cells inducing in them DNA fragmentation (73). TNFR1 can initiate several cellular responses, including apoptosis that relies on caspases and necrotic cell death, which depends on receptor-interacting protein kinase 1 (RIPK1; 74, 75). TRADD protein has been suggested to be a crucial signal adaptor that mediates all intracellular responses from TNFR1 (76). Caspase-2 is one of the earliest identified caspases engaged in the mitochondria-dependent apoptotic pathway by inducing the release of cytochrome c and other mitochondrial apoptogenic factors into the cell cytoplasm (77). PPM1F encodes a protein that is a member of the protein phosphatase 2C family of Ser/Thr protein phosphatases; overexpression of this phosphatase has been shown to mediate caspase-dependent apoptosis (78). Two apoptotic and two antiapoptotic genes are down-regulated in the breast epithelium of parous women: the programmed cell death 5 or PDCD5 and the transformed 3T3 cell double minute 4 (MDM4) in the former, and baculoviral inhibitor of apoptosis protein repeat–containing 6 (BIRC6) and BCL2-associated athanogene 4 (BAG4) in the latter. The Mdm4 gene that encodes structurally related oncoproteins that bind to the p53 tumor suppressor protein and inhibit p53 activity is amplified and overexpressed in a variety of human cancers (79). The Split hand/foot malformation (ectrodactyly) type 1 (SHFM1) encodes a protein with a BIR (baculoviral) domain and UBCc (ubiquitin-conjugating enzyme E2, catalytic) domain (80). This protein inhibits apoptosis by facilitating the degradation of apoptotic proteins by ubiquitination. BAG4 is a member of the BAG1-related antiapoptotic protein family that functions through interactions with a variety of cell apoptosis– and growth-related proteins including BCL2, Raf protein kinase, steroid hormone receptors, growth factor receptors, and members of the heat shock protein 70 kDa family. This protein was found to be associated with the death domain of TNF receptor type 1 and death receptor 3, and thereby negatively regulates downstream cell death signaling (81). Altogether these clusters of genes seem to maintain the programmed cell death pathway very active in the parous breast epithelium when compared with the epithelium obtained from the breast of parous women with cancer and from nulliparous women with or without cancer. Supporting evidence for this statement comes from data obtained from experimental models (6, 25) and from normal breast tissues of parous women obtained from reduction mammoplasties (6, 26), in which genes involved in the pathway of apoptosis are significantly deregulated. Another cluster of genes that are up-regulated in the parous control group are those related to immunosurveillance. We have previously reported that breast epithelial cells from parous women significantly overexpressed genes related to the immune system (82); therefore, this category will not be further discussed here.

Altogether, our data indicate that the first full-term pregnancy induces in the breast epithelium a specific genomic profile that is still identifiable in parous women at postmenopause. Furthermore, this genomic signature is constituted by genes that cluster differently than those genes expressed in the epithelial cells of parous and nulliparous women with breast cancer as well as from nulliparous women without cancer. This genomic signature allowed us to evaluate the degree of mammary gland differentiation induced by pregnancy. Of importance is the fact that this signature serves for characterizing at molecular level the fully differentiated condition of the breast epithelium that is associated with a reduction in breast cancer risk, thus providing a useful molecular tool for predicting when pregnancy has been protective, for identifying women at risk irrespective of their pregnancy history, and for its use as an intermediate biomarker for evaluating cancer preventive agents.

Grant support: National Cancer Institute grant RO1-CA093599.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Fox Chase Cancer Center Hospital Network Participants: Emily Penman and Nicholas J. Petrelli, Helen F. Graham Cancer Center, Christiana Care Health System, Newark, Delaware; Angela Lanfranchi, Somerset Medical Center, Somerville, New Jersey; Kathryn Evers, Diagnostic Imaging, and Monica Morrow, Department of Surgical Oncology, American Oncologic Hospital, Fox Chase Cancer Center, Philadelphia, Pennsylvania.

We thank Joanne F. Dorgan, M.P.H., Ph.D. (Genetic Epidemiology, Population Science Division, Fox Chase Cancer Center) for supervising the collection of breast tissues and of patient data; Debra Riordan, M.S., Ryan Hopson, M.S., and Irene Shandruk, M.S. (Genetic Epidemiology, Population Science Division, Fox Chase Cancer Center), Gladwyn Downes (Christiana Care Health System, Newark, DE), and Barbara Carney (Somerset Medical Center, Somerville, NJ) for collecting the clinical data of all the patients entered in this study; Peter Morrison (Department of Biostatistics, Biodiscovery, Inc., San Diego, CA) for his contribution in the analysis of cDNA microarray data; and Daniel A. Mailo, Rebecca C. Heulings, Patricia A. Russo, and Fathima S. Sheriff for their technical and administrative contributions for the successful completion of this work.

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