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* Lombardi Cancer Center, Department of Oncology, Georgetown University, Washington, DC and Department of Food Science and Human Nutrition, University of Illinois, Urbana, IL
2 To whom correspondence should be addressed. Email: clarkel{at}georgetown.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • Pregnancy • weight gain • mammary tumorigenesis • leptin • MAPK
Although pregnancy before age 20 y reduces life-time breast cancer risk, pregnancy after age 30 y has an opposite effect (1–3). It is assumed that the breasts of older women have acquired cells that have already undergone the first steps of neoplastic transformation, and high pregnancy hormone and growth factor levels enhance their growth (3). This is supported by findings showing that the higher the estrogen levels appear to be during pregnancy, the greater is an older mother's risk of developing breast cancer (4–9).
We found recently that excessive weight gain during pregnancy increased the mother's later breast cancer risk (10). The adipose tissue acts as an endocrine and metabolic organ, and thus influences the synthesis and bioavailability of estrogens and other sex steroids, insulin, insulin-like growth factor 1 (IGF-1)3 and adipokines, such as leptin and adiponectin (11,12). All of these hormones and growth factors are associated with breast cancer risk: adiponectin levels were inversely linked to breast cancer risk, whereas high levels of leptin, insulin, and IGF-1 were correlated with increased breast cancer risk (13–19). In the present study, we used leptin receptor (Ob-Rb) defective obese Zucker rats (20,21) to identify hormonal changes associated with excessive weight gain during pregnancy and to determine whether changes in pregnancy leptin levels are causally associated with increased breast cancer risk. Leptin was chosen because this hormone polypeptide is secreted mainly by adipose tissue, but it is also expressed in several other tissues, including the mammary epithelium (15,22). Cellular actions of leptin are initiated by leptin binding to specific receptors (mainly Ob-Rb), followed by the activation of STAT3 phosphorylation and the ras-dependent mitogen-activated protein (MAP)-kinase pathway (23).
To exclude the possibility that prepregnancy obesity influenced the data, we also included a group of Sprague-Dawley rats to the study. Because obese Zucker rats exhibit abnormal mating behavior and rarely become pregnant (24), we mimicked pregnancy in Zucker rats using hormonal exposures (25–27).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Genotyping. Heterozygous Zucker rats (lean phenotype) were bred in our animal facility; the offspring were genotyped using DNA extracted from the tails according to the NaOH method (29). PCR amplification was performed using primers described previously (21). After PCR amplification, products were submitted to restriction enzyme (Msp 1) digest and analyzed by electrophoresis in an agarose gel. Only homozygous (FA/FA) wild-type and homozygous mutant (fa/fa) Zucker rats were used in this study.
Experimental design. Sprague-Dawley rats were used to identify hormonal changes associated with excessive weight gain during pregnancy and to determine whether this excessive weight gain affected breast cancer risk. We then measured hormone levels associated with excessive weight gain during pregnancy, and determined mammary tumorigenesis between wild-type and mutant Zucker rats. Because the data were comparable in Sprague-Dawley and Zucker rats, only Zucker rats were used to attempt to identify the mechanisms that mediate the effects of excessive weight gain during pregnancy on dams' mammary tumorigenesis.
Carcinogen administration. At 50 d of age, Sprague-Dawley rats (n = 73), and lean (n = 77) and obese (n = 37) female Zucker rats were administered 10 mg of the mammary carcinogen 7,12-dimethylbenz[a]anthracene (DMBA; Sigma Chemical) by oral gavage. The carcinogen was dissolved in peanut oil and given in a volume of 1.0 mL. We administered the same dose of DMBA to all rats, regardless of the difference in body weight, to mimic human exposure to environmental carcinogens, which is independent of body weight.
Pregnancy manipulations. Two weeks after the DMBA exposure, Sprague-Dawley rats were mated (2 females and 1 male housed together), and hormonal exposures to mimic pregnancy were started in Zucker rats. These rats were implanted subcutaneously with silastic capsules containing 150 µg of estradiol (E2, Sigma Chemical) and 30 mg of progesterone (P; Sigma Chemical). Hormonal exposures lasted for 3 wk (length of pregnancy in rodents). On the day of mating, or a day after implantation of the E2 + P capsules, Sprague-Dawley rats and wild-type and mutant Zucker rats were assigned to 2 diet groups (Table 1) (1) control (16% energy from fat): Sprague-Dawley-control, lean-control and obese-control; and (2) obesity-inducing diet (45% energy from fat): Sprague-Dawley-OID, lean-OID and obese-OID. Diets were obtained from Harlan-Teklad. The high-fat diet (45% energy from fat) was found previously to elevate weight gain in rodents (30). When the pups were born or when the E2- and P- releasing capsules were removed, all rats were switched back to the AIN93G diet.
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10% of total body weight. All remaining rats, including those that did not develop tumors, were killed 25 wk after DMBA administration. Tumor histopathology classification was performed as previously described (31). Serum hormone and growth factor concentrations. Blood was collected by cardiac puncture from dams at d 10 and 17 of pregnancy (mimicked or real). Serum was separated and kept at –80°C until use. Levels of estradiol, progesterone, leptin, adiponectin, insulin, and IGF-1 were determined using rat enzyme immunoassay kits from Alpco Diagnostics (progesterone and estradiol), Assay Design, B-Bridge International, Linco Research, and Diagnostic Systems Laboratories, respectively, according to manufacturer instructions.
Mammary gland whole mounts and hyperplastic alveolar nodules (HAN) quantification. Only lean and obese Zucker rats were used to assess changes in mammary gland morphology, cell proliferation, or protein levels induced by an exposure to the OID during mimicked pregnancy. The mammary gland whole mounts used for these measurements, which were processed as previously described (32), were obtained from rats 25 wk after DMBA exposure. Whole mounts were evaluated under a light microscope to determine the number of hyperplastic alveolar nodules.
Cell proliferation. Mammary glands were fixed in 10% buffered formalin, embedded in paraffin, and sectioned (5 µm). Mammary gland sections were deparaffinized in xylene, hydrated through graded alcohols series, and then incubated with the primary antibody against PCNA at 1:500 dilution (Santa Cruz Biotechnology) overnight at 4°C, followed by incubation with the biotinylated anti-goat secondary antibody and avidin horseradish peroxidase complex (Vectastain Elite ABC Kit). PCNA staining was visualized by incubation with the chromogen 3,3'-diaminobenzidine (Vector Laboratories). The proliferation index was determined by calculating the percentage of cells that had positive PCNA staining (only dark stained cells were counted) in 1000 cells/mammary gland section.
Western blots. To determine activated MAPK protein levels, mammary glands were homogenized in buffer containing 25 mmol/L Tris-HCl, 2 mmol/L MgCl2, 1 mmol/L EDTA, 1% Triton X-100, 10% glycerol, 1 mmol/L dithiothreitol, 1 mmol/L phenylmethylsulfonyl fluoride, 0.001 mmol/L leupeptin, 0.001 mmol/L pepstatin, and 1 mmol/L aprotinin. Lysates were separated on a NuPAGE 12% Bis-Tris gel (Invitrogen Life Technologies) and blotted onto nitrocellulose. Membranes were blocked in 5% milk for 30 min at room temperature. Membranes were then incubated with phospho-MAPK Thr202/Tyr 204 antibodies (1:1000 dilution, Cell Signaling Technology) overnight at 4°C. Next, membranes were washed with Tris-Buffered Saline Tween-20 (TBST) and incubated with the secondary anti-rabbit IgG horseradish peroxidase antibody (1:5000, Amersham Pharmacia Biotech) at room temperature for 1 h. Membranes were washed with TBST and developed using electrochemiluminescence (Amersham Pharmacia Biotech). For total MAPK levels, blots were stripped, washed several times with TBST, and reblocked. Membranes were then incubated with primary antibody for total MAPK (1:1000, Cell Signaling), followed by incubation with the secondary antibody (1:5000), as described above. Fold differences were calculated by normalizing phosphorylated MAPK to total MAPK.
Statistical Analysis.
The following statistical tests were utilized to analyze the data: 1) Student's t test (body weight at the beginning and end of pregnancy, and pregnancy leptin, estradiol, and IGF-1 levels on gestation d 17 in Sprague-Dawley rats); and 2) 2-way ANOVA (body weight at the beginning and end of mimicked pregnancy in Zucker rats, leptin, estradiol, IGF-1, insulin, and adiponectin levels on gestation d 17, number of HAN, and activation of MAPK in Zucker rats using pregnancy dietary exposure and genetic background as variables, estradiol and progesterone levels on gestation d 10 or 17 between control diet fed pregnant Zucker rats and Zucker rats that underwent mimicked pregnancy using pregnancy and genetic background as variables). Data of pregnancy hormone concentrations, mammary tumorigenesis, cell proliferation, and MAPK activation were also obtained from a group of pregnant lean Zucker rats fed a control diet; these data were for comparison purposes only and were not included in the statistical analysis. Because the estrous cycle may influence mammary cell proliferation in rats, the proliferation index data were analyzed by 2-way ANOVA, using the stage of estrous cycle and pregnancy dietary exposure as independent variables. Between-group comparisons were done using Tukey's test. Tumor incidence was calculated using the methods developed by Kaplan and Meier, followed by the log-rank test. Differences in final tumor incidence among groups were compared using the 
2-test. All tests were performed using SPSS SigmaStat software; differences were considered significant when P < 0.05. All probabilities were 2-tailed.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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To determine possible changes in hormone concentrations induced by excessive weight gain during pregnancy, serum estradiol, leptin, and IGF-1 levels were measured in rats on gestation d 17. Leptin concentrations were significantly higher in pregnant Sprague-Dawley rats fed the OID (1.9 ± 0.16 nmol/L) than in rats fed the control diet (1.3 ± 0.15 nmol/L) (P < 0.037), but the concentrations of estradiol and IGF-1 were not affected. Among the Zucker rats, leptin concentrations were higher in the obese than in the lean rats (P < 0.001), and the OID increased leptin levels in both groups (P < 0.029) (Table 3). Concentrations of adiponectin (P < 0.01) and insulin (P < 0.001) were higher in the obese Zucker rats compared with the lean Zucker rats, but IGF-1 concentrations did not differ. Exposure to the OID did not modify concentrations of adiponectin, insulin, or IGF-1 in the Zucker rats.
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2 times that of the controls (Table 4). Mammary tumor multiplicity, latency, or tumor growth did not differ between the groups.
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Histological evaluation of mammary tumors revealed that most of the tumors were adenocarcinomas. Further, we examined mammary gland whole mounts prepared from the 4th abnominal glands to determine whether Zucker rats also developed preneoplastic mammary lesions. The OID did not induce hyperplastic alveolar nodules (HAN), but the number of HAN was higher in the mammary glands of obese Zucker rats compared with the glands of wild-type rats (P < 0.001) (Fig. 2A, B).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In addition to the increase in breast cancer risk that occurred when the rats were exposed to the OID during pregnancy, obese Zucker rats (both OID- and control diet–fed) had a significantly higher mammary tumor incidence after the mimicked pregnancy than lean Zucker rats, in agreement with a recent study showing that nulliparous obese Zucker rats have a significantly higher mammary tumor incidence than lean rats (33). However, the results are in contrast with a study by Lee et al. (34) who noted that lean and obese Zucker rats exposed to NMU had similar rates of mammary tumor development. The reason for the different findings may be that in the study of Lee et al. (34), obese Zucker rats received 
of the dose of NMU given to lean rats even though they were significantly heavier. In the present study, all rats were administered the same dose of DMBA and in the study of Hakkak et al. (33), the DMBA dose was adjusted to body weight.
Pregnancy weight gain was strongly associated with increased leptin levels in Sprague-Dawley rats and in both the lean and obese Zucker dams. This is in line with a previous observation that the amount of adipose tissue is linked directly to the amount of leptin in the circulation (35), also during pregnancy (36). However, if an increase in leptin levels was associated with increased breast cancer risk, then a minimal increase in leptin signaling is sufficient to affect breast cancer risk. Leptin binds to its receptor with the same affinity in obese and lean Zucker rats, but the defective receptor in obese Zucker rats effectively inhibits most of the leptin receptor–induced signaling (37–39). However, we cannot exclude the possibility that some activation takes place and that this activation is sufficient to affect promotion of breast cancer.
The serum concentrations of estradiol and IGF-I were unaffected in the Sprague-Dawley or Zucker rat dams fed the OID, consistent with studies in humans showing that body weight during pregnancy does not correlate with estradiol levels (40). Further, the evidence linking circulating IGF-1 levels to obesity is controversial (41). Two other hormones also were determined during mimicked pregnancy in Zucker rats: insulin and adiponectin. Neither hormone was altered by exposure to a high-fat diet, although they were higher in the obese Zucker rats than in the lean rats. Hyperinsulenemia is positively associated with breast cancer risk in women (42,43), and according to a recent observation, breast cancer risk is increased in women with gestational glycosuria (44), i.e., these women had high circulating levels of insulin during pregnancy. In agreement with these reports, we showed here that obese Zucker rats had high levels of circulating insulin during mimicked pregnancy and they developed more mammary tumors than their lean counterparts. Adiponectin levels inversely correlate with body weight (35), but in the present study, the levels were higher in obese than lean Zucker rats. Because adiponectin levels are regulated by several factors, including estrogens (45,46), the high pregnancy hormonal environment may be responsible for the disparity between the present study done in "pregnant" dams and the previous studies that used nonpregnant rats.
Several mechanisms may link hyperisulenemia to breast cancer, including insulin's mitogenic effects (47). Consistent with these effects, the proliferation index in the mammary glands of obese Zucker rats was higher than that observed in the mammary glands of lean dams. In addition, activation of MAPK, a downstream target of the insulin pathway that is strongly linked to cell proliferation, was markedly increased in the mammary glands of obese compared with lean rats. Increased signaling through the MAPK pathway was linked to breast cancer (48); for example, activation of MAPK is increased in 50–100% of breast cancers (49,50). Further, a recent proteomics study of mammary adipose tissue of breast cancer patients showed that the MAPK pathway, among other signaling pathways, is activated in adipose tissues surrounding the tumor (51). Importantly, although increased insulin signaling might be associated with a high breast cancer risk in obese Zucker rats, it does not appear to explain the effects of excessive weight gain during pregnancy on later breast cancer risk: neither pregnancy insulin levels nor cell proliferation in the mammary gland were associated with an exposure to the OID during mimicked pregnancy. MAPK activity was lower in the mammary glands of rats fed the OID, suggesting that exposure to a high-fat diet during mimicked pregnancy caused a long-lasting reduction in this signaling pathway.
The present study, a combined analysis of 3 different rat genetic backgrounds, showed that as in humans (10), excessive weight gain during pregnancy increased dams' mammary tumorigenesis. The only hormone that was associated with excessive weight gain during pregnancy was leptin, but because the OID-fed leptin receptor–defective Zucker rats also exhibited increased breast carcinogenesis, the role of the leptin receptor in mediating the observed effects remains to be established. Further, in future studies, we will attempt to identify some other biological factor(s) not measured here, i.e., factors other than estradiol, IGF-1, insulin, or adiponectin that might mediate the effects of excessive weight gain during pregnancy on mother's breast cancer risk.
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FOOTNOTES |
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3 Abbreviations used: DMBA, 7,12-dimethylbenz(a)anthracene; E2, estradiol; fatty, fa/fa; HAN, hyperplastic alveolar nodule IGF-1, insulin-like growth factor 1; MAPK, mitogen-activated protein kinase; NMU, N-methyl-M-nitrosourea; Ob-Rb, leptin receptor; OID, obesity-inducing diet; P, progesterone.
Manuscript received 16 August 2005. Initial review completed 1 September 2005. Revision accepted 13 January 2006.
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