Exposure to Excess Estradiol or Leptin during Pregnancy Increases Mammary Cancer Risk and Prevents Parity-Induced Protective Genomic Changes in Rats

Pregnancy affects a woman's breast cancer risk by first inducing a transient increase in risk, lasting for 5 to 7 years (1–4), and then either permanently reducing or increasing the risk, depending upon the age of the woman. Women who gave birth before age 20 decrease their breast cancer risk by half compared to women who were over 30 when they had their first child (5). The latter, in turn, have a significantly higher lifetime risk of breast cancer than nulliparous women (6, 7). The protective effect of early pregnancy is limited to estrogen and progesterone receptor positive (ER+ and PR+) breast cancers (8, 9), whereas late first pregnancy can increase the risk of developing either ER+ or ER− cancers (9, 10).

Several theories have been offered to explain the protective effects of early pregnancy on breast cancer risk (11, 12). Importantly, parous women and animals exhibit permanent changes in gene expression patterns, resulting in a pregnancy-induced protective genomic signature. This signature involves genes that can prevent malignant transformation, including those that reduce mammary epithelial cell proliferation and increase differentiation (13–15). It is less clear why a late first pregnancy increases breast cancer risk, but it may be caused by an aging-related increase in the presence of transformed mammary epithelial cells that can start proliferating when exposed to a high pregnancy hormonal environment. Accumulating evidence indicates that women who had the highest circulating estrogen levels during pregnancy (16, 17) or were exposed to the synthetic estrogen diethylstilbestrol (DES; refs. 18 and 19) are at highest risk of developing breast cancer. In addition, giving birth to an infant with high birth weight is associated with a high maternal estriol/α-fetoprotein ratio and increased breast cancer risk (20).

The possibility that elevated leptin levels during pregnancy also may increase breast cancer risk has not been explored. Serum leptin concentrations increase during pregnancy, peaking during the second trimester (21, 22), although the increase is not nearly as dramatic as with estrogens. Pregnant women who gain an excessive amount of weight have high leptin levels (23–25) and are significantly more likely to develop breast cancer after menopause than women whose weight gain during pregnancy does not exceed the recommendations provided by the Institute of Medicine (IOM; ref. 26). In preclinical studies, excessive weight gain induced by feeding pregnant dams an obesity-inducing high-fat diet increases pregnancy leptin levels and subsequent mammary tumorigenesis (27). Importantly, leptin interacts with estradiol (E2) and the ER. Leptin has been shown to activate ER-alpha, likely through its ability to stimulate aromatase and/or mitogen-activated protein kinases (MAPK; refs. 28 and 29). Furthermore, leptin decreases ER-alpha ubiquitination and increases ER-alpha half-life, potentially leading to increased ER-alpha activity (30). E2, in turn, can interfere with leptin's actions by regulating the expression of the leptin receptor (31). Similar to E2, leptin promotes the growth of ER+ human breast cancer cells in culture (32, 33), but it also induces proliferation of ER− breast cancer cells (34).

In this study, we sought to establish experimentally whether treating pregnant dams with excess E2 or leptin during pregnancy increases later mammary tumorigenesis in rats. Our results indicate that in the vehicle-treated control rats pregnancy induced a transient increase in mammary cancer risk that lasted until mammary glands had undergone involution and returned to a nonpregnant and nonlactating stage. When back to this stage, the risk of developing breast cancer was dramatically reduced, resulting in a lower lifetime risk than what was seen in nulliparous rats. Rats exposed to an excess of either E2 or leptin during pregnancy exhibited a sustained increase in mammary tumorigenesis, similar to nulliparous rats. Higher breast cancer risk in the parous E2 or leptin rats than in vehicle-treated parous control rats may be related to a persistent increase in cell proliferation in their mammary glands, and absence of parity-induced protective changes in the genome. Thus, our preclinical study suggests that an exposure to excess E2 or leptin during pregnancy increases risk by preventing pregnancy-induced reduction in breast cancer risk and the protective changes in genomic signaling pathways seen in the parous mammary gland.

Five-week-old Sprague-Dawley rats were obtained from Charles River and fed AIN93G diet upon arrival. Animals were housed in a temperature- and humidity-controlled room at the Georgetown University Resource Animal Facility under a 12-hour light–dark cycle. All animal procedures were approved by the Georgetown University Animal Care and Use Committee, and the experiments were performed following the National Institutes of Health guidelines for the proper and humane use of animals in biomedical research.

At 50 days of age, a total of 223 female rats were administered 10 mg of the mammary carcinogen 7,12-dimethylbenz(a)anthracene (DMBA; Sigma Chemical Co.) by oral gavage. Carcinogen was dissolved in peanut oil and given in a volume of 1 mL.

Two weeks after DMBA exposure, female rats were mated by housing 2 female rats and 1 male rat together. Positive vaginal plug was used to determine the first day of pregnancy. On gestation day 8, pregnant females were divided into 3 experimental groups: control dams receiving subcutaneous vehicle injections (n = 43), E2 dams receiving subcutaneous injections of 10 μg of 17β-estradiol (Sigma Chemical Co.; n = 42), or leptin dams receiving subcutaneous injections of 15 μg of leptin (R&D systems; n = 40). Injections were given daily until gestation day 19. The doses were chosen based upon a pilot study that indicated that neither 10 μg E2 nor 15 μg leptin affected weight development during pregnancy. After giving birth, dams were allowed to nurse their offspring for 3 weeks, and then the pups were weaned.

An additional set of 78 DMBA-exposed female rats, 3 weeks after the carcinogen exposure (to match with day 8 of gestation), were divided to 3 groups and given subcutaneous injections of vehicle (n = 29), 10 μg of E2 (n = 41), or 15 μg of leptin (n = 28). Injections were given daily for a total of 2 weeks.

Four weeks post-DMBA administration, we began checking rats weekly for mammary tumors by palpation. Tumor growth was measured using a caliper and the length, width, and height of each tumor were recorded. Animals were sacrificed if any tumor reached a size of 25 to 30 mm in diameter. The remaining animals, including those that did not develop tumors, were sacrificed 17 weeks after pregnancy ended/22 weeks after DMBA administration. Endpoints for this study were time to tumor appearance (tumor latency), the number of tumors per animal (tumor multiplicity), and the percentage of rats that developed tumors per experimental group (tumor incidence).

Concentrations of circulating leptin and E2 were determined in serum collected by tail bleeding on gestation day 19 (n = 5–7 per group), using a rodent leptin EIA kit from Assay Designs Inc. and a rodent E2 EIA kit from Cayman Chemical Company, respectively, following the manufacturers' instructions.

At the end of the tumor-monitoring period (22 weeks post-DMBA exposure), all rats were sacrificed and their mammary tissues and tumors were obtained. Cell proliferation in the mammary tissue was assessed by immunohistochemistry staining for PCNA in 6 rats per group. The second and third glands were used and they were fixed in 10% buffered formalin, embedded in paraffin, and sectioned (5 μm). Sections were deparaffinized in xylene, hydrated through graded alcohols, and incubated with 3% H₂O₂ for 10 minutes to block endogenous peroxidases. Nonspecific binding was blocked with normal rabbit serum from the Vectastain Elite ABC Kit (Vector Laboratories, Inc.) for 20 minutes. Tissue sections were incubated overnight with the primary antibody against PCNA at a 1:500 dilution (Santa Cruz Biotechnology Inc.). After several washes, sections were treated with the secondary antibody (biotinylated anti-goat IgG from the Vectastain Elite ABC Kit; Vector Laboratories, Inc.) for 30 minutes at room temperature, followed by treatment with an avidin and biotinylated horseradish peroxidase complex from the Vectastain Elite ABC Kit (Vector Laboratories, Inc.) for 30 minutes at room temperature. Sections were washed and stained with 3,3′-diaminobenzidine (DAB; Vector Laboratories, Inc.) for 1 minute, washed, and counterstained with Vector's Hematoxylin QS Nuclear Counterstain (Vector Laboratories, Inc.) for 45 seconds. Proliferation index was determined by calculating the percentage of cells with positive PCNA staining in at least 1,000 cells per mammary gland section. Slides were evaluated using the Metamorph software, without knowledge of treatment group.

Apoptosis was assessed in the same mammary gland sections used to determine proliferation (n = 6 per group) by in situ oligo ligation (ISOL) assay with an ApopTag Kit (Serologicals Corporation) following the manufacturer's instructions. Briefly, sections were deparaffinized in xylene and hydrated in a series of graded alcohols. The sections were then treated with 20 μg/mL of Proteinase K for 15 minutes. Endogenous peroxidases were quenched with 3% H₂O₂ for 5 minutes. Sections were washed with equilibration buffer (ApopTag Kit) and incubated with the Ligase enzyme for 16 hours at 16°C to 22°C. The reaction was stopped and sections were incubated with a streptavidin–peroxidase conjugate at room temperature. Sections were again washed, incubated with the peroxidase substrate for 10 minutes, and counterstained with 0.5% methyl green (Vector Laboratories, Inc.) for 10 minutes. Apoptotic index was determined by calculating the percentage of cells that were apoptotic through both positive staining and histologic evaluation among 1,000 cells per mammary gland section. All sections were evaluated using the Metamorph software, without knowledge of treatment group.

The fourth mammary glands that contained no palpable growth or nonpalpable microtumors were obtained from 5 rats per group (control, E2, and leptin exposed), sacrificed 22 weeks after DMBA exposure. Six micrograms of purified total RNA was used to synthesize cDNA and then generate cRNA, which was labeled with biotin according to techniques recommended by Affymetrix. Labeled cRNA was fragmented at 94°C for 35 minutes in a fragmentation buffer and then hybridized to Affymetrix Rat U34 A GeneChips, which contained approximately 7,000 full-length sequences and 1,000 EST clusters. After washing, the chips were stained with strepavidin–phycoerythrin conjugate and then scanned using the Affymetrix GeneChip Scanner 3000 (Hewllet-Packard Co.). Raw data were generated using Affimetrix GeneChip 3.1 software.

In Affimetrix GeneChip experiments, variations in the amount and quality of target hybridized to the array may contribute to an overall variability in hybridization intensities. To reliably compare data from multiple probe arrays, differences of nonbiological origin must be minimized. We accomplished this by normalizing the data using the MicroArray Suite 5.0 (Affymetrix) software to average the intensities for each GeneChip and to calculate a normalization factor. The normalized intensities were obtained from each chip by multiplying raw intensities by the normalization factor.

Normalized results obtained from each group were used to calculate the ratio (control/treated) for each gene. Hybridization signal intensities of relative fold changes, which ranged from ≤0.5 for downregulation or ≥2-fold for upregulation, were considered to be significant and were reported. The level of significance was set at P < 0.05. Dimensionality reduction (elimination of noninformative data) was performed by filtering out genes with low threshold (intensity < 0.1 in both groups) and low fold change (<2.0). In addition, comparisons made had to be significantly different in at least 1 of 3 statistical tests (i.e., equal and unequal variance t tests, equal and unequal variance t tests on log transformed data, Wilcoxon test).

We calculated the 3-dimensional projections of multidimensional gene expression microarray datasets by using principal component analyses and discriminant component analyses.

To determine whether the model could accurately predict the leptin/E2 exposure, a neural network was trained, independent of gene expression profile selection.

Quantitative real-time PCR (qRT-PCR) was used to confirm the differential expression of selected genes between the control and high-risk groups shown in the microarray analysis. The 4th mammary glands were obtained from a different set of rats (n = 6–8 per group) than the ones used for microarray analysis. Briefly, cDNA was reverse transcribed from 50 μg/mL of total input RNA using Taqman Reverse Transcription Reagents (Applied Biosystems). The reverse transcription reaction was carried out in a Taqman master mix under the following conditions: 25°C for 10 minutes, 48°C for 30 minutes, and 95°C for 5 minutes. Next, PCR products were generated from the cDNA samples using the Taqman Universal PCR Master Mix (Applied Biosystems) and Assays-on-Demand (Applied Biosystems) for the appropriate target gene (Vegfa, Pleiotrophin, Mapk 9, and Eif4e). The 18S Assay-on-Demand (Applied Biosystems) was used as an endogenous control in all assays. All assays were run on 384-well plates so that each cDNA sample was run in triplicate for the target gene and the endogenous control. qRT-PCR was performed on an ABI Prism 7900 Sequence Detection System and the results assessed by relative quantification of gene expression using the ΔΔC_T method.

Data for pregnancy hormone levels and gene expression were analyzed by ANOVA (only assessed in parous rats). Some mammary tumor endpoints (latency and multiplicity) were analyzed by two-way ANOVA, using nulliparous or parous, and treatments as independent variables. Cell proliferation and apoptosis were only assessed in parous rats, and because the estrous cycle may influence mammary cell proliferation and apoptosis in rats (low proliferation: pro-estrus, estrus, and the second part of diestrus; and high proliferation: metestrus and the first part of diestrus), the proliferation and apoptosis indices data were analyzed by two-way ANOVA, using the stage of estrous cycle and E2/leptin exposure as independent variables. Where appropriate, comparisons between groups were done using Holm–Sidak method. Kaplan–Meier curves were used to compare differences in tumor incidence, followed by the log-rank test. Tumor incidence was also analyzed just for post-DMBA weeks 13 and 22, and in this analysis nulliparous control rats were compared to parous control rats and either parous rats exposed to leptin or E2 were included in the analysis. Final tumor incidence was determined using χ² test. All tests were performed using the SPSS SigmaStat software, and differences were considered significant if the P-value was less than 0.05. All probabilities were two-tailed.

Neither E2 nor leptin affected weight gain during pregnancy (Table 1). Birth weights of the pups also were similar, as were the numbers of pups born per litter (Table 1). The concentrations of circulating E2 and leptin, measured in serum samples collected on day 19 of pregnancy, are shown in Fig. 1. Leptin levels were significantly higher in the leptin-exposed dams when compared to either the E2 or control dams (P < 0.001). Circulating E2 levels were significantly higher in the E2-treated group, when compared to the control or leptin-treated dams (P = 0.004).

Because pregnancy has a transient and long-term effect on breast cancer risk, we considered tumors that developed between weeks 1 and 12 after DMBA as early appearing tumors, and those developing on week 13 or after as long term. Twelve weeks post-DMBA treatment coincided with completion of mammary gland involution in parous rats, as the rats became pregnant 2 weeks after DMBA, gave birth 5 weeks after DMBA and started undergoing involution 8 weeks after DMBA. It then takes 4 weeks for the rat mammary gland to return to a prepregnancy stage (13, 35); that is, this occurred on week 12 in our study.

We first determined whether a 2-week exposure of nulliparous rats to E2 or leptin alters mammary tumorigenesis. Table 2 indicates that the mean mammary tumor latency in nulliparous control rats is about 13 weeks. Tumor latency did not differ among the nulliparous control-, E2-, or leptin-exposed rats. In the vehicle-treated nulliparous rats, 41% of the tumors become palpable during weeks 1 and 12, and 59% during weeks 13 to 22. The majority of the tumors in the leptin group (79%) were detected before week 13, whereas in the E2 group 23% of the tumors were detected early and 77% were detected after week 12 (P < 0.004). Final mammary tumor incidence and multiplicity were similar in the 3 groups of nulliparous rats exposed to vehicle, leptin, or E2. These results are shown in Table 2 and Fig. 2A.

Next, we compared mammary tumorigenesis in the vehicle (control)-treated nulliparous and parous rats. Latency of mammary tumor appearance was shorter in the parous than nulliparous rats (P < 0.005). In the parous control rats, 93% of the tumors appeared during weeks 1 and 12, compared to 41% in the nulliparous group (P < 0.001). The final tumor incidence during weeks 1 and 22 (P < 0.001) and during weeks 13 and 22 (P < 0.001) in the nulliparous controls was higher than in the parous rats, but tumor multiplicity was similar (Table 2). Thus, similar to women, we found that after a transient increase in mammary cancer risk, pregnancy provided protection against breast cancer in rats.

In the parous control group, all but one (7%) of 14 tumors became palpable within 12 weeks of DMBA exposure, whereas 12 (46%) of the 26 tumors in the leptin group and 10 (53%) of the 19 tumors in the E2 group appeared after week 12 of pregnancy (P < 0.018; Table 2). This is similar to what was seen in nulliparous control rats in which 59% developed mammary tumors after week 12. Thus, although the mean tumor latency period was longer in both the leptin (P < 0.001) and E2-treated parous rats (P < 0.002) than in the vehicle-treated parous rats, it did not differ between the parous hormone-treated rats and nulliparous control rats; that is, the treatments did not delay tumor development.

To determine whether an exposure to leptin or E2 during pregnancy affected mammary tumorigenesis, differences were assessed between weeks 13 and 22. Both the leptin (P < 0.001) and E2 groups (P < 0.0037) exhibited significantly higher mammary tumor incidence than the parous control rats (Fig. 2), but neither group differed from nulliparous control rats. At the end of the monitoring period, final tumor incidence was higher in the parous rats exposed during pregnancy to either leptin (65%) or E2 (45%), when compared to the controls (33%) (Table 2), but this difference reached statistical significance in the leptin group only (P < 0.039). However, tumor multiplicity among the groups was not statistically different (Table 2).

Cell proliferation and apoptosis were determined in mammary glands obtained from rats sacrificed 22 weeks after exposure to DMBA. Figure 3 shows that the proliferation index, determined by PCNA staining, was significantly higher in the mammary glands of E2-treated parous rats compared to those of vehicle-treated parous control rats (P < 0.001). The number of apoptotic cells present in the mammary glands of rats in the 2 treatment groups and controls were determined using the ISOL assay. There were no significant differences among these 2 treatment groups, when compared to the controls (P = 0.17; Fig. 4).

To explore the long-term effects on gene expression in the mammary glands of rats exposed to E2 or leptin during pregnancy, microarray experiments were performed using RNA extracted from mammary glands collected 22 weeks after DMBA exposure. In the comparison between the control and leptin groups, 352 genes were found to be differentially expressed (criteria for differential expression was 2-fold difference and P < 0.05). The comparison between the control and E2 groups revealed 252 differentially expressed genes. We then compared the E2 and leptin groups, and found only 11 genes to be differentially expressed between these 2 groups. For this reason, these 2 groups were combined into 1 high-risk group and compared to controls. In this analysis, we identified 143 genes associated with changes in tumorigenesis between the control and high-risk groups. Of those, 62 genes were downregulated (Table 3) and 80 genes upregulated (Table 4) in the high-risk group compared to controls.

Several of the genes that were differentially expressed in the mammary glands of parous rats exposed to either leptin or E2 during pregnancy, compared to controls, are involved in cell growth, survival, and angiogenesis. These genes included Mapk9 (mitogen-activated protein kinase 9), Nras (neuroblastoma ras oncogene), Ptn (pleiotrophin), Vegfa (vascular endothelial growth factor), and Eif4e (eukaryotic initiation translation factor 4e), which were upregulated in the mammary gland of rats exposed to leptin or E2 during pregnancy when compared to vehicle treated controls (Table 4). We also found that the expression of genes inducing mammary epithelial differentiation, such as α-lactalbumin and α-casein, were downregulated in the leptin- or E2-exposed dams (Table 3).

Differential expression of these genes was confirmed by real-time PCR. As illustrated in Fig. 5, transcripts for Vegfa and Ptn were more abundant in the rats treated with either leptin or E2 during pregnancy than in the controls (P < 0.001 and P < 0.001, respectively). Vegfa levels were 3.8- and 6.8-fold higher in mammary glands of leptin and E2-treated dams than in the controls, respectively (Fig. 5A). Ptn mRNA levels were 3.3-fold higher in leptin-treated and 21-fold higher in E2-treated dams than in the controls (Fig. 5B).

Real-time PCR data indicated that the levels of Mapk9 mRNA were 1.3-fold higher in the leptin-treated parous rats than in the vehicle- or E2-treated dams (Fig. 5C; P = 0.008). Transcription levels of Eif4e were 1.2-fold higher in mammary glands of leptin-treated animals compared to the controls (P = 0.003; Fig. 5D). Differential expression of Nras in the microarray was not confirmed by real-time PCR.

Several earlier studies have outlined a gene expression signature characterizing the effect of parity on the mammary gland. We investigated whether there were any similarities between these signatures and changes in gene expression induced by an exposure to excess leptin or E2 during pregnancy. For that purpose, we used the tables of differentially expressed genes between parous and nulliparous rat and mouse strains generated in studies by D'Cruz and colleagues (13) and Blakely and colleagues (14), and humans by Asztalos and colleagues (36).

Several common genes in the parous rats exposed to E2 or leptin versus vehicle, and parous versus nulliparous animals and women were identified. The genes identified in this comparison are shown in Tables 5 and 6. Importantly, genes that were upregulated (or downregulated) in parous rats, compared to nulliparous rats, were also upregulated (or downregulated) in vehicle-treated parous rats, compared to parous rats treated with E2 or leptin during pregnancy, suggesting that these hormonal exposures prevented parous-induced signaling changes in the mammary glands. For example, TGF-β3 has been reported to be upregulated in parous animals and humans, compared to nulliparous controls, and we found that is was also upregulated in parous control rats, compared to parous rats treated with leptin or E2 during pregnancy. The downregulated genes are those that induce differentiation (Csn1, Cp, and Lalba) or regulate immune functions (Lcn2 and Lbp), whereas the upregulated genes are those that promote growth (Ghr and Ptn), angiogenesis (Vegfa) and induce epithelial-to-mesenchymal transition (Col1a1).

Only one gene, Cited 1, was found to be altered in a similar manner both in the parous animals (compared to nulliparous animals) and in the leptin- or E2-exposed parous rats (compared to control parous rats) in our study. Cited 1 is a transcriptional coregulator of ER-α and affects estrogen sensitivity in a gene-specific manner (37). Therefore, pregnancy suppresses ER-α signaling, with increasing suppression the higher the hormone levels were during pregnancy. However, we did not observe any changes in the expression of ER-α between the parous rats which received E2 or leptin during pregnancy and parous control rats. Instead, G protein–coupled estrogen receptor 1 (Gper) that localizes to the endoplasmic reticulum and binds estrogen was downregulated in the parous E2- and leptin-treated rats. This protein is involved in the rapid nongenomic signaling events observed with estrogen.

Results obtained in our study indicate that parous control rats had a lower mammary tumor incidence than nulliparous rats which is consistent with the protective effect of pregnancy against breast cancer in women who have their first child before age 20 (5) and previous reports in rats (27, 38). Importantly, the majority of mammary tumors in parous rats in our study appeared before mammary gland involution had been completed. These findings are in accordance with the transient increase in breast cancer risk caused by pregnancy in women (1–4). An exposure to E2 or leptin during pregnancy increased mammary cancer risk in parous rats. Specifically, E2- or leptin-treated parous rats continued to develop mammary tumors also after the initial transient increase in risk. Thus, the pattern of mammary tumor development in the rats treated with E2 or leptin during pregnancy mimicked that of nulliparous rats, suggesting that the hormonal exposures prevented the protective effects of parity on mammary cancer risk.

The increase in mammary cancer risk in rats exposed to E2 or leptin during pregnancy is consistent with findings reported in humans. Women who took the synthetic estrogen DES during pregnancy are at an increased risk of developing breast cancer (18, 19). Furthermore, women who exhibit the highest pregnancy estrogen levels, either in the first (16) or third (17) trimesters of gestation are at elevated breast cancer risk later in life. We are not aware of any studies that have investigated whether leptin levels during pregnancy affect later breast cancer risk among mothers, but indirect parameters of high leptin levels, such as obesity or weight gain (23–25) indicate that parous women who had the highest leptin levels during pregnancy also are at an increased risk of developing breast cancer. Excessive weight gain during pregnancy is common: close to 50% of pregnant women gain more than recommended by the IOM (26, 39). Because these women are at an increased risk of developing breast cancer after menopause (26), the results obtained in our animal model suggest that high leptin levels during pregnancy are responsible, at least partly, for this finding.

The mechanisms responsible for the association between elevated E2 or leptin levels during pregnancy and increased breast cancer risk remain to be elucidated. We performed microarray analysis to identify differentially expressed genes in the mammary glands between the parous control rats and parous rats exposed to E2 or leptin during pregnancy. Intriguingly, only 11 (0.05%) of >7,000 genes were differentially expressed between the rats that were exposed to E2 or leptin during pregnancy, although both groups exhibited a number of differentially expressed genes compared to controls. The similarity of gene expression in the 2 hormone-treated groups may reflect the close association between leptin and estrogen signaling in the mammary gland (28–31, 33). We therefore focused on the 142 differentially expressed genes, shown in Table 4, between the mammary glands of rats exposed to vehicle or E2/leptin during pregnancy.

The differentially expressed genes included Eif4e, Mapk9, Nras, Ptn, and Vegfa. All these genes have been linked to breast cancer. Deregulation of protein synthesis is a hallmark of many cancers, and overexpression of eukaryotic translation factor Eif4e contributes to the deregulation. It is overexpressed in breast cancers and high expression is linked to an elevated risk of recurrence (40). When overexpressed, Eif4e may enable the translation of a select pool of mRNAs encoding for proteins involved in malignant growth, such as those for cyclin D1, c-MYC, VEGF, and matrix metalloprotease-9 (MMP-9; ref. 41). Mapk9 regulates cell proliferation and apoptosis (42) and inhibition of its activity reduces cell proliferation in breast cancer cells (43). Ptn is overexpressed in at least 60% of human breast cancers (43), and this overexpression is linked to high risk of metastasis (44). Vegfa is often upregulated in breast tumors, especially in those expressing HER-2/neu (45) or mutant p53 (46). Furthermore, both leptin and estrogens activate Vegfa (47, 48). Leptin itself can induce angiogenesis in vitro and in vivo (49), and a neutralizing anti-leptin receptor monoclonal antibody suppresses leukemia cell growth by inhibiting angiogenesis in rats (50). Thus, we were able to confirm upregulation of Eif4e, Mapk9, Ptn, and Vegfa in the mammary glands of parous rats exposed to leptin or E2 during pregnancy, compared to parous control rats, and these changes may be associated with their increased mammary tumorigenesis. Increase in Nras expression in the microarray analysis was not confirmed by qRT-PCR.

In addition to these genes, several others were differentially expressed between control and E2/leptin-exposed parous rats. We were particularly interested in those genes that have been suggested to explain the protective effect(s) of pregnancy in rodents (13, 14) and humans (36). Thirteen of them were identified as differentially expressed between the parous control and E2/leptin-exposed rats. Importantly, genes that have been reported to be upregulated in the parous women/rodents, compared with nulliparous women/rodents, were upregulated in the parous control rats in our study, compared to parous rats treated with E2 or leptin during pregnancy. Thus, gene expression patterns in the E2/leptin-treated parous rats resembled those in the nulliparous rats. The same applied to downregulated genes: those that are found to be downregulated in parous versus nulliparous women/rodents were downregulated in parous control rats, compared to parous rats treated with E2 or leptin during pregnancy. Most of the downregulated genes (that are upregulated by parity) in the mammary glands of parous rats treated with E2 or leptin during pregnancy were those that induce differentiation (Csn1, Cp, and Lalba), inhibit growth (Tgfβ3), or regulate immune functions (Lcn2, and Lbp). Among the upregulated genes in the parous E2/leptin rats (and downregulated in parous women and rodents) were Vegfa, Ghr and Ptn that promote growth, and Col1a1 that induces cancer progression by stimulating epithelial mesenchymal transition. Our findings suggest that high levels of E2 and leptin during pregnancy may prevent parity-induced reduction in breast cancer risk by preventing protective signaling changes in mammary gland.

The parity-induced protective signaling patterns are likely to induce or reflect functional changes that result in reduced breast cancer risk. During pregnancy, the mammary gland undergoes substantial morphological changes, but after the gland has involuted, it returns to a stage resembling that seen in nulliparous animals (51, 52) or humans (12, 53). Findings in mice suggest that pregnancy promotes functional differentiation at a cellular level, and causes a reduction in the proportion of mammary epithelial stem/progenitor cells and an increase in differentiated luminal and myoepithelial cells (54, 55). Because breast cancer is thought to be initiated in epithelial stem/progenitor cells or differentiated cells that acquire stem cell like properties (56), reduction in stem/progenitor cell population may explain why early pregnancy reduces later breast cancer risk. In our study, parous rats exposed to E2 or leptin during pregnancy exhibited a sustained increase in cell proliferation, compared with parous control rats. Proliferating cells represent a progenitor cell population (57), and thus it is possible that a high hormonal environment during pregnancy prevents a pregnancy-induced reduction in stem cells. Although we did not determine whether there were less differentiated cells in the mammary glands of rats treated with E2 or leptin during pregnancy than in the controls, microarray analysis indicated that several genes that induce differentiation were downregulated, and those increasing cell proliferation were upregulated (Tables 5 and 6). In addition to the ones already discussed earlier, these included downregulation of Keratin 19, which is a marker of differentiated luminal cells (58).

In our study, parous rats treated with E2 or leptin during pregnancy exhibited higher mammary cancer risk than parous control rats, consistent with the findings in humans showing that women exposed to DES (19), having the highest pregnancy E2 levels (16, 17), or gaining more weight during pregnancy than recommended by the IOM (26), are at an increased risk of developing breast cancer. Parous control rats exhibited a transient increase in mammary cancer risk that lasted until their mammary gland had completed involution. After this transient period, the risk of developing mammary cancer was very low. However, in the parous rats treated with E2 or leptin during pregnancy the risk of developing mammary tumors remained elevated past the transient increase. Thus, the pattern of mammary tumor appearance in the parous E2- and leptin-exposed rats was similar to that seen in nulliparous rats, suggesting that parity does not protect against breast cancer if the levels of E2 or leptin during pregnancy are excessive. This conclusion is supported by comparing the pregnancy-induced protective mRNA signature obtained in earlier microarray analyses in rodents and humans (13, 14, 36) to the signature in parous rats treated with E2 or leptin during pregnancy. Gene expression in the mammary glands of E2- or leptin-treated parous rats was similar to that of nulliparous individuals. Thus, an exposure to excess E2 or leptin during pregnancy prevents the protective effects of pregnancy on the mammary gland and increases subsequent breast cancer risk. These findings suggest that pregnant women should avoid being exposed to the highest levels of E2 and leptin during pregnancy, caused for example by gaining excessive amounts of weight during pregnancy, because it may not only put them at risk of developing gestational diabetes and hypertension (59), but also increase later breast cancer risk.

No potential conflicts of interest were disclosed.

Conception and design: L.A. Hilakivi-Clarke

Development of methodology: L.A. Hilakivi-Clarke

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. de Assis, L.A. Hilakivi-Clarke

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. de Assis, M. Wang, L. Jin

Writing, review, and/or revision of the manuscript: S. de Assis, L. Jin, K. Bouker, L.A. Hilakivi-Clarke

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. de Assis, K. Bouker, L.A. Hilakivi-Clarke

Study supervision: L.A. Hilakivi-Clarke

This study was supported by National Cancer Institute (1P30-CA51008, R01 89950, 1R01CA164384, U54 CA000970, and U54 CA149147) for L. A. Hilakivi-Clarke.

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.