Insulin-like growth factor (IGF)-I obliterates the pregnancy-associated protection against mammary carcinogenesis in rats: evidence that IGF-I enhances cancer progression through estrogen receptor-α activation via the mitogen-activated protein kinase pathway

bcr812 BCJ Research article Insulin-like growth factor (IGF)-I obliterates the pregnancy-associated protection against mammary carcinogenesis in rats: evidence that IGF-I enhances cancer progression through estrogen receptor-α activation via the mitogen-activated protein kinase pathway Thordarson Gudmundur gummi@biology.ucsc.edu Slusher Nicole slusher@scripps.edu Leong Harriet thehairyit@yahoo.com Ochoa Dafne Rajkumar Lakshmanaswamy rajkumar@uclink4.berkeley.edu Guzman Raphael nandilab@uclink4.berkeley.edu Nandi Satyabrata nandilab@uclink4.berkeley.edu Talamantes Frank talamantes@biology.ucsc.edu

Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, California, USA

Cancer Research Laboratory, University of California, Berkeley, California, USA

Breast Cancer Res 1465-5411 2004 6 4 R423 R436 http://breast-cancer-research.com/content/6/4/R423 1521751110.1186/bcr812 22 3 2004 28 4 2004 8 5 2004 12 5 2004 4 6 2004 2004 Thordardson et al.; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. estrogen receptor-α growth hormone/insulin-like growth factor-I mammary carcinogenesis mitogen-activated protein kinase progesterone receptor

Abstract

Introduction

Pregnancy protects against breast cancer development in humans and rats. Parous rats have persistently reduced circulating levels of growth hormone, which may affect the activity of the growth hormone/insulin-like growth factor (IGF)-I axis. We investigated the effects of IGF-I on parity-associated protection against mammary cancer.

Methods

Three groups of rats were evaluated in the present study: IGF-I-treated parous rats; parous rats that did not receive IGF-I treatment; and age-matched virgin animals, which also did not receive IGF-I treatment. Approximately 60 days after N-methyl-N-nitrosourea injection, IGF-I treatment was discontinued and all of the animal groups were implanted with a silastic capsule containing 17β-estradiol and progesterone. The 17β-estradiol plus progesterone treatment continued for 135 days, after which the animals were killed.

Results

IGF-I treatment of parous rats increased mammary tumor incidence to 83%, as compared with 16% in parous rats treated with 17β-estradiol plus progesterone only. Tumor incidence and average number of tumors per animal did not differ between IGF-I-treated parous rats and age-matched virgin rats. At the time of N-methyl-N-nitrosourea exposure, DNA content was lowest but the α-lactalbumin concentration highest in the mammary glands of untreated parous rats in comparison with age-matched virgin and IGF-I-treated parous rats. The protein levels of estrogen receptor-α in the mammary gland was significantly higher in the age-matched virgin animals than in untreated parous and IGF-I-treated parous rats. Phosphorylation (activation) of the extracellular signal-regulated kinase-1/2 (ERK1/2) and expression of the progesterone receptor were both increased in IGF-I-treated parous rats, as compared with those in untreated parous and age-matched virgin rats. Expressions of cyclin D₁and transforming growth factor-β₃in the mammary gland were lower in the age-matched virgin rats than in the untreated parous and IGF-I-treated parous rats.

Conclusion

We argue that tumor initiation (transformation and fixation of mutations) may be similar in parous and age-matched virgin animals, suggesting that the main differences in tumor formation lie in differences in tumor progression caused by the altered hormonal environment associated with parity. Furthermore, we provide evidence supporting the notion that tumor growth promotion seen in IGF-I-treated parous rats is caused by activation of estrogen receptor-α via the Raf/Ras/mitogen-activated protein kinase cascade.

Introduction

A first full-term pregnancy early in life confers effective natural protection against breast cancer in women 1. Rats exhibit a similar phenomenon, in that parity prevents chemically induced mammary carcinogenesis 2. The causes of this pregnancy-associated protection against mammary carcinogenesis are still being investigated. Changes in the mammary epithelia, such as high degree of differentiation, low level of proliferation, increase in cell cycle length, reduction in carcinogen binding to epithelial cells, and increased capacity for DNA repair have been associated with parity in rats 34567. More recently it was shown that gene expression is altered in the mammary gland of parous mice and rats as compared with virgin animals 8; similarly, rats that have been made refractory to mammary tumorigenesis by estrogen and progesterone treatments also exhibit differences in the mammary gland gene expression as compared with untreated rats 9. However, the functional significance of these alterations in gene expression in relation to the susceptibility of the mammary gland to carcinogenesis has not been demonstrated.

Parity also causes changes in the circulating levels of hormones that regulate mammary gland development and may affect the susceptibility of the mammary gland to tumorigenesis. For example, parity in women causes a persistent reduction in the concentration of prolactin in serum 1011, and similarly parous rats have a significantly reduced circulating concentration of growth hormone (GH) as compared with nulliparous, age-matched animals 2. Furthermore, in our previous studies 12 we demonstrated that treatment of parous rats with low doses of 17β-estradiol and progesterone abolishes the protective effects induced by pregnancy. Therefore, the pregnancy-associated protection against mammary cancer can be nullified by changing the hormonal environment of the animal.

These findings cast doubts upon whether the changes in the mammary epithelia of parous animals are permanent phenotypical alterations or a reflection of altered hormonal environment. As mentioned above, we found a reduction in the circulating concentration of GH associated with parity in rats. An increasing body of evidence now indicates that the GH/insulin-like growth factor (IGF)-I axis is a determining factor in the susceptibility of the breast to cancer development. Early studies showed that administration of GH together with estrogen and progesterone restores chemically induced mammary tumorigenesis in hypophysectomized rats 13. Later studies showed that inhibition of GH secretion reduces chemically induced mammary carcinogenesis in rats 1415. Similarly, mice carrying a transgene expressing GH antagonist (modified bovine GH) are at reduced risk for developing mammary cancer as compared with littermates not carrying the transgene 16; in contrast, mice overexpressing GH exhibit an increase in mammary cancer development as compared with mice with normal GH levels 17.

Direct effects of IGF-I on normal mammary gland development and mammary carcinogenesis are also evident. IGF-I-deficient (knockout) mice exhibit very limited mammary epithelial development 18; this defect was remedied by IGF-I and 17β-estradiol treatment, whereas 17β-estradiol treatment alone was ineffective. Overexpression of IGF-I transgene targeted to the mammary gland by placing it under the control of whey acidic protein promoter inhibits involution of the mammary epithelia after lactation, indicating that IGF-I acts as a survival factor for the mammary epithelia 1920. This inhibition of involution is acquired, at least partly, through reduction in apoptosis of mammary cells 1921. Furthermore, continuous breeding of mice carrying the whey acidic protein promoter-regulated IGF-I transgene results in mammary cancer development, albeit after a long latency period 21.

Epidemiologic studies have shown that a high circulating concentration of IGF-I is correlated with increased risk for breast cancer development 22, and GH and IGF-I concentrations in serum are elevated in breast cancer patients 2324. How the GH/IGF-I axis affects mammary tumorigenesis is not well established. The mitogenic activity of IGF-I in normal mammary epithelia is well known 25, and GH has been shown to regulate estrogen receptor (ER) expression in the mammary gland 26. Indeed, we have found a significant reduction in levels of ER in mammary gland of parous rats as compared with age-matched virgin animals 2. In the present study we investigated the effects of IGF-I treatment on the parity-associated protection against mammary cancer.

Methods

Animals

Female Sprague–Dawley rats, 6–15 per group, were kept in a 14-hour light/10-hour dark lighting schedule, and were given free access to food and water. To generate parous animals, virgin rats were mated at 50–55 days of age. After parturition, the pups were removed and the mammary glands of the mothers were allowed to involute for 40 days. At that time, the parous rats were used for experimentation. N-methyl-N-nitrosourea (MNU) was purchased from Ashe Stevens (Detroit, MI, USA) and administered in a single intraperitoneal injection 27. Three groups of rats were used in the experiments. In one group, parous rats were treated with recombinant human IGF-I (Genentech Inc., South San Francisco, CA, USA) at a dose of 0.660 mg/kg body weight per day, administered via an Alzet osmotic pump (Durect, Cupertino, CA, USA), for 60 days commencing 7 days before MNU injection and continued for an additional 53 days. The second group included parous rats that did not receive any IGF-I treatment. The third group included age-matched virgin control animals, which also did not receive any IGF-I treatment. Approximately 60 days after the MNU injection, 1 week after the IGF-I treatment was terminated, all animals were implanted with a silastic capsule 28 containing 20 μg 17β-estradiol (Sigma, St Louis, MO, USA) and 20 mg progesterone (Sigma). The capsules were changed every 2 months. The 17β-estradiol plus progesterone treatment was continued for 135 days, after which all animals were killed. To assess the development of the mammary gland and the serum concentrations of IGF-I at the time of carcinogen administration, animals from each of the three groups were killed 7 days after commencement of treatment. Normal mammary tissues and serum were collected and stored at -80°C until they were analyzed (Fig. 1).

Figure 1

Schematic representation of the animal treatments used in the present study

Schematic representation of the animal treatments used in the present study. E2, 17β-estradiol; IGF, insulin-like growth factor; MNU, N-methyl-N-nitrosourea; P4, progesterone.

Wholemounts were prepared from the right second and third glands from the animals and used for assessment of mammary development. The frozen mammary tissues were used for Western blot analyses as described below and to assess total DNA, α-lactalbumin, and IGF-I contents of the mammary gland.

The MNU-treated animals were palpated weekly for detection of mammary tumors and tumors were removed from the animals under anesthesia when they had grown to 1.5 cm in diameter. At the time of tumor collection, a small sample was excised from each tumor for histologic classification. The serum samples were used to measure the circulating concentrations of IGF-I at the time of MNU injection.

The care and use of animals in the study was approved by the Chancellor's Animal Care Committee at the University of California at Santa Cruz.

Assessment of α-lactalbumin in mammary tissues

The content of α-lactalbumin in the mammary tissues was used as an indicator of differentiation of the mammary epithelia at the time of carcinogen exposure. The tissues were ground to a fine powder in liquid nitrogen with pestle and mortar and then homogenized on ice with a Polytron homogenizer (Brinkmann Instruments Inc., Burlingame, CA, USA) in 2 volumes (weight/vol) of 50 mmol/l Tris-HCl, 5 mmol/l MgCl₂buffer (pH 7.5) containing 1 mmol/l Pefabloc (Roche Molecular Biochemicals, Indianapolis, IN, USA) and 1 μmol/l Pepstatin A (Sigma). After the tissues had been homogenized, 20 μl samples were obtained and used for measuring the total DNA content of the preparation, and the remainder of the samples were extracted for 1 hour and then centrifuged at 20,000 g for 30 min; both steps were conducted at 4°C. The supernatant was collected and used to measure total protein and α-lactalbumin concentration. A radioimmunoassay, specific for rat α-lactalbumin, was developed. Rat α-lactalbumin was purified from rat milk in accordance with a previously established method 29. Antiserum generated against rat α-lactalbumin was generously provided by Dr Kurt E Ebner at the University of Kansas Medical Center. The within and between coefficient variations of the assay were 3.0% and 18.4%, respectively.

Wholemount preparation and tumor histology

Mammary gland wholemounts were prepared as described previously 30. Briefly, animals were killed and pinned down on a corkboard, ventral side up. A skin incision was made between the nipples and down both the hind legs, creating a cut in the shape of an inverted 'Y'. The mammary glands were exposed, and the right second and third glands removed and fixed in 10% buffered formalin. After de-fatting the tissue in acetone, the mammary epithelia was stained with iron/hematoxylin and inspected under a microscope for assessment of overall development. All tumors were classified microscopically. For that, a small specimen was obtained, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. The sections were then stained with hematoxylin and eosin and classified.

Measurement of IGF-I concentration in mammary tissues

IGF-I was extracted from the mammary glands as has previously been described 31. Briefly, the tissues were pulverized as described above, homogenized in 1 N acetic acid (1 g tissue/5 ml acetic acid), and extracted on ice for 2 hours. After centrifugation at 20,000 g for 6 min, the supernatant was collected and the tissues extracted again as before, and the two extractions for each sample were combined and lyophilized. The lyophilized material was reconstituted in 50 mmol/l Tris/HCl (pH 7.8) at 1 g original wet weight of tissue per 2 ml buffer and assayed using radioimmunoassays for rat and, for those animals treated with human IGF-I, for human IGF-I (Diagnostic Systems Laboratories, Webster, TX, USA) either undiluted or diluted in assay buffer.

Protein and DNA assays

The serum concentrations of endogenous IGF-I and exogenous IGF-I (human IGF-I in treated rats) were assessed using radioimmunoassay kits from Diagnostic Systems Laboratories, as described above. The protein concentrations of extracted mammary samples were measured with BCA protein assay kit (Pierce, Rockford, IL, USA) using bovine serum albumin (Sigma) as the reference standard. The total DNA content of the mammary gland homogenates was assessed by fluorometric DNA assay 29 using calf thymus DNA (Sigma) as reference standard.

Western blot analyses

Western blot analyses were carried out as recently described 32. Briefly, mammary gland tissues were homogenized and extracted, and solubilized protein electrophorized on 7.5–20% SDS-PAGE, depending on the size of the protein being analyzed, at 50–100 μg total protein/lane, as determined by BCA protein assay (Pierce). Proteins were transferred to a PVDF membrane and the specific protein bands detected using chemiluminescence reagents and CL-X Posure™ Film (Pierce). Western blot analyses were carried out on cyclin D₁using antibody sc-450 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); total ER-α using antibody Ab-15 (NeoMarkers Inc., Fremont, CA, USA); progesterone receptor (PR) using antibody no. A 0098 (DakoCytomation Inc., Carpinteria, CA, USA); total and phosphorylated extracellular signal-regulated kinase-1/2 (ERK1/2) using antibodies #9102 and #9106, respectively (Cell Signaling Technology Inc., Beverly, MA, USA); and transforming growth factor (TGF)-β₃using antibody GF16 (Oncogene Research Product, San Diego, CA, USA). Protein bands, detected with chemiluminescence, were quantified using the ImageJ (version 1.24o) image analysis program (National Institutes of Health, Bethesda, MD, USA).

Statistics

Incidence of mammary cancer was analyzed using 2 × 2 contingency tables and χ²tests. All other statistical analyses were carried out using analysis of variance and Fisher's protected least significant difference test. P < 0.05 was considered statistically significant.

Results

Treatment of parous rats with 0.66 mg IGF-I/kg body weight per day resulted in an increase in the circulating concentration of total IGF-I to 2289.2 ± 68.8 ng/ml (mean ± standard error), which was a significantly higher concentration than that found in intact parous and age-matched virgin rats (Fig. 2a). The IGF-I concentration in the mammary tissues at the time of carcinogen exposure was also elevated in the IGF-I-treated parous animals as compared with untreated parous rats, but did not differ significantly between IGF-I-treated parous and age-matched virgin animals (Fig. 2b). Body weight gain did not differ between the animal groups, when assessed at the time of MNU injection and at the termination of the experiment (Table 1). However, the elevation of IGF-I in parous rats caused a significant increase in mammary tumor incidence as compared with parous rats treated only with 17β-estradiol plus progesterone, beginning 60 days after the MNU injection. Tumor incidence in IGF-I-treated parous rats and age-matched virgin rats that also received 17β-estradiol and progesterone treatment 60 days after MNU injection did not differ significantly, and neither did the average number of tumors per animal in these two groups (Table 2). All the animals that did carry mammary tumors developed at least one carcinoma (mostly ductal, papillary, and cribriform carcinomas). Development of fibroadenoma was rare (only 6–7%) and did not differ between IGF-I-treated parous and age-matched virgin rats.

Figure 2

Insulin-like growth factor (IGF)-I concentration in (a) serum and (b) mammary tissues of parous rats treated with insulin-like growth factor (IGF)-I (P-IGF-I), untreated parous rats (P-Un), and age-matched virgin rats (AMV). IGF-I treatment (0.66 mg/kg body weight/day) was continued for 7 days before samples were collected. IGF-I concentrations for P-IGF-I rats are combined endogenous (rat IGF-I) and exogenous (human IGF-I) values. Values are expressed as mean ± standard error. *P < 0.05: versus AMV and P-Un in panel a, and versus P-Un in panel b.

Table 1

Body weight

Groups

Body weight (g) at MNU injection (mean ± SEM)

Body Wt. (g) at Sacrifice (mean ± SEM)

P-Un

286 ± 3.2 (n = 7)

303 ± 3.5 (n = 6)

P-IGF-I

286 ± 6.2 (n = 7)

317 ± 4.5 (n = 6)

AMV

293 ± 5.4 (n = 7)

309 ± 9.6 (n = 6)

Body weight at the time of N-methyl-N-nitrosourea (MNU) injection and just before the animals were killed at the termination of the experiment for untreated parous rats (P-Un), parous rats treated with insulin-like growth factor-I (P-IGF-I), and untreated virgin rats that were age matched with the parous animals (AMV).

Table 2

Mammary carcinogenesis in N-methyl-N-nitrosourea injected rats

Groups

Cancer incidence (%)

Cancer load number/rat (mean ± SEM)

Latency range (days)

P-UN (n = 6)

16*

0.167

121

P-IGF-I (n = 6)

2.17 ± 0.75

57–176

AMV (n = 6)

100

2.20 ± 0.45

92–217

Mammary tumor incidence, latency, and load in untreated parous rats (P-Un), parous rats treated with insulin-like growth factor -I, and in virgin rats that were age matched with the parous animals (AMV). *P < 0.05 versus P-IGF-I and AMV.

The total DNA content of the mammary glands was significantly lower in the parous rats that did not receive IGF-I treatment, as compared with IGF-I-treated parous rats and age-matched virgin animals (Fig. 3). The stage of differentiation, as measured using the content of α-lactalbumin in the mammary gland, was significantly higher in mammary tissues from untreated parous rats compared with IGF-I-treated parous and age-matched virgin rats (Fig. 4). Inspection of the whole mounts revealed that the epithelial structures were less dense in the parous untreated rats than in the parous IGF-I-treated and particularly in the age-matched virgin rats. Also apparent from the inspection of the whole mounts was that terminal end-buds (TEBs) were present in all of the animal groups and did not appear to be less abundant in the parous untreated animals than in age-matched virgin and IGF-I-treated parous rats (Fig. 5). These structures are traditionally associated with a high rate of epithelial proliferation and high susceptibility to cancer development.

Figure 3

The total DNA content of mammary glands of parous rats treated with insulin-like growth factor (IGF)-I (P-IGF-I), untreated parous rats (P-Un), and age-matched virgin rats (AMV)

The total DNA content of mammary glands of parous rats treated with insulin-like growth factor (IGF)-I (P-IGF-I), untreated parous rats (P-Un), and age-matched virgin rats (AMV). The IGF-I treatment (0.66 mg/kg body weight/day) was continued for 7 days before samples were collected. Values are expressed as mean ± standard error. *P < 0.05 versus AMV and P-IGF-I.

Figure 4

The concentration of α-lactalbumin (α-lac) in mammary tissues obtained from parous rats treated with insulin-like growth factor (IGF)-I (P-IGF-I), untreated parous rats (P-Un), and age-matched virgin rats (AMV). The IGF-I treatment (0.66 mg/kg body weight/day) was continued for 7 days before samples were collected. Values are expressed as mean ± standard error. *P < 0.05 versus AMV and P-IGF-I.

Figure 5

Photomicrographs showing mammary gland wholemounts from parous rats treated with insulin-like growth factor (IGF)-I (upper row, panel a; lower row, panel a), untreated parous rats (upper row, panel b; lower row, panel b), and age-matched virgin rats (upper row, panel c; lower row, panel c). The IGF-I treatment (0.66 mg/kg body weight/day) was continued for 7 days before the animals were killed and the wholemounts were prepared. Note the smaller alveolar structures of the mammary epithelia from parous animals, particularly untreated parous rats, as compared with the age-matched virgin rats (upper row of panels) and the presence of terminal end-buds in the mammary glands from all groups (lower row of panels). Magnifications: 6.25× (upper row) and 10× (lower row).

The total protein level of ER-α in the mammary gland at the time of carcinogen injection was significantly higher in age-matched virgin animals than in untreated parous rats, and treatment of parous rats with IGF-I further lowered the concentration of ER-α (Fig. 6). Phosphorylation of ERK1/2 was significantly increased in mammary tissues of IGF-I-treated parous rats, whereas the lowest level of phosphorylation of ERK1/2 was found in the mammary tissues from untreated parous rats (Fig. 7). No difference was found in the levels of total ERK1/2 expression in mammary glands from the three groups of rats (Fig. 8). Like phosphorylation of ERK1/2, the expression of PR in mammary tissues was significantly elevated in animals treated with IGF-I, but PR expression was lowest in untreated parous animals (Fig. 9).

Figure 6

Western blot analysis showing estrogen receptor (ER)-α expression in mammary gland extracts from parous rats treated with insulin-like growth factor (IGF)-I (P-IGF-I), untreated parous rats (P-Un), and age-matched virgin rats (AMV). The IGF-I treatment (0.66 mg/kg body weight/day) was continued for 7 days before samples were collected. The protein samples (100 μg/lane) were electrophorized on 7.5% SDS-PAGE, transferred to PVDF membrane, and probed with antibody specific to ER-α(Ab-15; NeoMarkers Inc., Fremont, CA, USA). Protein bands were detected using enhanced chemiluminescence reagents (upper image) and quantified using the ImageJ (version 1.24o; National Institutes of Health, Bethesda, MD, USA) image analysis program (bar chart). Values are expressed as mean ± standard error. *P < 0.05 versus P-IGF-I and P-Un.

Figure 7

Western blot analysis showing the level of phosphorylation of extracellular signal-regulated kinase-1/2 (pERK1/2) in mammary tissues from parous rats treated with insulin-like growth factor (IGF)-I (P-IGF-I), untreated parous rats (P-Un), and age-matched virgin rats (AMV). The IGF-I treatment (0.66 mg/kg body weight/day) was continued for 7 days before samples were collected. The protein samples (50 μg/lane) were electrophorized on 10% SDS-PAGE, transferred to PVDF membrane, and probed with antibody generated to phosphorylated form of human ERK1/2 (#9106; Cell Signaling Technology Inc., Beverly, MA, USA). Protein bands were detected using enhanced chemiluminescence reagents (upper image) and quantified using the ImageJ (version 1.24o; National Institutes of Health, Bethesda, MD, USA) image analysis program (bar chart). Values are expressed as mean ± standard error. *P < 0.05 versus P-Un and AMV.

Figure 8

Western blot analysis showing the level of total extracellular signal-regulated kinase-1/2 (ERK1/2) in mammary tissues from parous rats treated with insulin-like growth factor (IGF)-I (P-IGF-I), untreated parous rats (P-Un), and age-matched virgin rats (AMV). The IGF-I treatment (0.66 mg/kg body weight/day) was continued for 7 days before samples were collected. The protein samples (50 μg/lane) were electrophorized on 10% SDS-PAGE, transferred to PVDF membrane, and probed with antibody generated to rat ERK1/2 (#9102; Cell Signaling Technology Inc., Beverly, MA, USA). Protein bands were detected using enhanced chemiluminescence reagents (upper image) and quantified with the ImageJ (version 1.24o; National Institutes of Health, Bethesda, MD, USA) image analysis program (bar chart). Values are expressed as mean ± standard error.

Figure 9

Levels of progesterone receptor (PR) in mammary glands obtained from parous rats treated with insulin-like growth factor (IGF)-I (P-IGF-I), untreated parous rats (P-Un), and age-matched virgin rats (AMV). The IGF-I treatment (0.66 mg/kg body weight/day) was continued for 7 days before samples were collected. Protein samples (50 μg/lane) were electrophorized on a 7.5% SDS-PAGE; Western blot analysis was carried out using a specific anti-PR antibody (no. A 0098; DakoCytomation Inc., Carpinteria, CA, USA) and specific protein bands detected using enhanced chemiluminescence reagents. The upper image shows the results of the Western blot analysis and the bar chart shows the quantitation and statistical analysis of the results. Values are expressed as mean ± standard error. *P < 0.05 versus P-Un and AMV.

Expression of cyclin D₁was lowest in mammary gland from age-matched virgin animals but was similar in mammary tissues from the two parous groups (Fig. 10). Similarly, the levels of TGF-β₃were found to be lowest in mammary glands from age-matched virgin rats, but TGF-β₃did not differ in mammary tissues obtained from the untreated and IGF-I-treated parous animals (Fig. 11).

Figure 10

The expression of cyclin D₁in the mammary gland of parous rats treated with insulin-like growth factor (IGF)-I (P-IGF-I), untreated parous rats (P-Un), and age-matched virgin rats (AMV)

The expression of cyclin D₁in the mammary gland of parous rats treated with insulin-like growth factor (IGF)-I (P-IGF-I), untreated parous rats (P-Un), and age-matched virgin rats (AMV). The IGF-I treatment (0.66 mg/kg body weight/day) was continued for 7 days before samples were collected. Protein samples (100 μg/lane) were electrophorized on a 10% SDS-PAGE, transferred to a PVDF membrane, and specific protein bands detected using a specific cyclin D₁antibody (sc-450; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and enhanced chemiluminescence reagents. The upper image shows the results of the Western blot analysis and the bar chart shows the quantitation and statistical analysis of the results. Values are expressed as mean ± standard error. *P < 0.05 versus P-Un and P-IGF-I.

Figure 11

Levels of transforming growth factor (TGF)-β₃in mammary glands obtained from parous rats treated with insulin-like grwoth factor (IGF)-I (P-IGF-I), untreated parous rats (P-Un), and age-matched virgin rats (AMV)

Levels of transforming growth factor (TGF)-β₃in mammary glands obtained from parous rats treated with insulin-like growth factor (IGF)-I (P-IGF-I), untreated parous rats (P-Un), and age-matched virgin rats (AMV). The IGF-I treatment (0.66 mg/kg body weight/day) was continued for 7 days before samples were collected. Protein samples (100 μg/lane) were electrophorized on a 20% SDS-PAGE; Western blot analysis was carried out using a specific anti-TGF-β₃antibody (GF16; Oncogene Research Product, San Diego, CA, USA) and specific protein bands detected using enhanced chemiluminescence reagents. The upper image shows the results of the Western blot analysis and the bar chart shows the quantitation and statistical analysis of the results. Values are expressed as mean ± standard error. *P < 0.05 versus P-Un and P-IGF-I.

Discussion

Several years ago we found that the circulating levels of GH and, to a lesser extent, of prolactin are significantly reduced in parous rats, and we speculated that this reduction in serum concentration of GH might be a determining factor in the reduced susceptibility of the mammary gland to cancer development associated with parity 2. Much other evidence links GH and/or IGF-I (the GH/IGF-I axis) with both normal mammary gland development 2533 and possible involvement in carcinogenesis of the breast 3435. In the present study we demonstrated that IGF-I treatment increases mammary tumorigenesis in parous rats to a level similar to that in age-matched virgin animals. We also previously showed that long-term treatments of parous rats with low doses of 17β-estradiol and progesterone obliterate the parity-associated protection against mammary cancer 12. In addition, it has been shown that dissociated mammary epithelial cells obtained from MNU-treated young virgin rats develop fewer tumors and exhibit a longer latency period when transplanted into parous syngeneic hosts as compared with cells transplanted into virgin syngeneic hosts 36.

The prevailing hypothesis regarding how parity protects the breast against cancer development has been that the mammary epithelia consist of undifferentiated, fast-growing TEB and terminal duct structures that are highly susceptible to carcinogenesis, and of differentiated, slow-growing alveolar structures that exhibit refractoriness to cancer development. Extensive differentiation of the mammary gland seen at parturition rids the gland of the fast-growing susceptible TEBs and terminal ducts, replacing them with the differentiated, refractory alveolar structures. Accompanying the reduction in the proliferation rate of the mammary epithelia of the parous animals are other changes, such as increased capacity for DNA repair, decreased binding of the carcinogen to the DNA, and increased length of cell cycle.

According to this hypothesis, this condition of the mammary gland is retained after involution of the gland; that is, differentiation of the mammary gland acquired during pregnancy is a permanent state 34567. It is now clear, based on a number of studies, that this hypothesis inadequately explains the differences in susceptibility of mammary gland to tumorigenesis between virgin and parous animals. First, it has long been questioned whether there is much difference in the proliferative activity of the mammary gland of virgin as compared with parous rats. In terms of structure, TEBs are as abundant in the mammary gland of parous as they are in virgin rats 1237, which we confirmed in the present study. Assessment of proliferation confirms findings from the structural studies. Using thymidine incorporation, Sinha and coworkers 37 did not find any difference in labeling index between mammary glands from parous and age-matched virgin animals at the time of carcinogen exposure.

However, this study and previous reports support the notion that the mammary gland maintains higher levels of differentiation, at least in terms of milk-specific protein expression, after pregnancy or hormonal treatment that causes pregnancy-like development of the gland, as compared with mammary glands of virgin, intact animals 8912. Nevertheless, it has been difficult to demonstrate an association between a previous differentiated state of the mammary gland and its subsequent susceptibility to tumorigenesis. For example, stimulating the development of the mammary gland almost to a lactational state by increasing the circulating levels of prolactin and progesterone without changing the 17β-estradiol concentration in serum does not confer protection of the gland against MNU-induced carcinogenesis, whereas treatment of the animals with 17β-estradiol either alone or with progesterone does provide protection 38. Also, Rajkumar and coworkers 39 were unable to establish a good correlation between mammary differentiation and 17β-estradiol-induced protection against tumorigenesis, in that full protection was conferred after a short-term estrogen treatment without full lobule–alveolar development. Similarly, Medina and coworkers 40 found a discrepancy between mammary development and the level of protection using low doses of estrogen and progesterone, and termination of pregnancy before any significant differentiation of the mammary gland has taken place confers partial protection against mammary tumorigenesis 37. Further refuting the notion that differentiation of the mammary gland is a prerequisite for refractoriness to tumorigenesis, and supporting the claim that the hormonal environment is the determining factor, is the finding that virgin Sprague–Dawley dwarf rats lacking functional GH, caused by a point mutation of the gh gene 41, exhibit the same refractoriness to mammary tumorigenesis as normal, parous Sprague–Dawley rats 4243.

As mentioned above, we reported earlier 2 that the circulating concentrations of both GH and prolactin are reduced in parous rats as compared with age-matched virgin rats. Importantly, it has now been demonstrated that short-term treatment of virgin rats with 17β-estradiol alone or in combination with progesterone, to achieve circulating levels of these hormones comparable to those seen in late pregnant animals, confers mammary tumor refractoriness and significant reduction in the circulating concentrations of GH and prolactin 44. Furthermore, GH-deficient Sprague–Dawley dwarf rats exhibit the same refractoriness to mammary tumorigenesis that is seen in parous rats 4243, but when treated with GH the dwarf rats acquire the same high susceptibility seen in normal virgin Sprague–Dawley rats 43. Also, in the present study we show that the same increase in mammary tumorigenesis can be achieved by treating the parous rats with IGF-I. Therefore, it is unequivocal that the activity of the GH/IGF-I axis is fundamental in determining the level of mammary carcinogenesis. However, the question remains as to how the hormonal environment affects the mammary gland to increase or decrease its tumorigenesis. Will the answer be found in a difference in expression of specific genes in the mammary gland at the time of carcinogen exposure, causing an increase/decrease in transformation upon carcinogen exposure?

Using DNA microarrays, D'Cruz and coworkers 8 identified a number of genes that were differentially expressed in mammary glands from parous and virgin rats and mice. Similarly, Ginger and coworkers 9 used a subtractive suppressive hybridization method to analyze the differences in gene expression of the mammary gland from susceptible (intact virgin) and refractory (estrogen and progesterone treated) Wistar–Furth rats. Again, a number of genes were found to be differentially expressed in the susceptible and refractory glands, but it remains to be seen whether any of these differentially expressed genes are involved in determining the susceptibility of the mammary gland to tumor development.

We studied here the expression of a few specific genes that we considered likely to be important for the susceptibility of the mammary gland to carcinogenesis. However, we found it difficult to relate an increase or a decrease in gene expression to an increase or a decrease in tumorigenesis of the mammary gland at the time of carcinogen exposure. For example, we found the lowest expression of the ER-α in mammary tissues from IGF-I-treated parous rats, but mammary tumorigenesis in these animals was the same as in age-matched virgin rats. The same difficulties are encountered when interpreting the results for expression levels of cyclin D₁and TGF-β₃. Cyclin D₁, which is a cell cycle regulator 45 and causes mammary cancer when overexpressed, as evident from studies in the cyclin D₁transgenic mouse model 46, did not correlate well with tumorigenesis, with the lowest expression occurring in animals with the highest tumorigenesis (age-matched virgin rats). D'Cruz and coworkers 8 previously showed that cyclin D₁mRNA levels are increased in the mammary gland of parous rats as compared with age-matched virgin animals. Also, as D'Cruz and coworkers reported, we found the lowest level of TGF-β₃expression in mammary tissues of age-matched virgin rats, which is a possible indication of a high proliferation rate 47, but no difference was found in mammary tissues obtained from untreated and IGF-I-treated parous animals, although tumorigenesis in these two parous groups differs significantly.

It should also be considered in this context that there might actually not be much, if any, difference in susceptibility to tumor initiation (transformation and fixation of mutation) between mammary glands of parous and virgin animals. It was recently demonstrated that tumor formation does occur in the mammary gland of parous rats, with incidence rate and multiplicity similar to that in age-matched virgin rats 48. However, these tumors stay largely latent at microscopic size and grow only to a palpable size upon hormonal stimulation, as we demonstrated here and showed in previous studies 12. That is, tumor formation does occur in the mammary epithelia of parous rats, but the hormonal environment of these animals is altered to the extent that it is not capable of promoting tumor progression. Supporting the notion that the difference in palpable mammary tumor development between parous and virgin animals lies primarily in the promotion of growth, but not in transformation, is the fact that the protection – conferred either by pregnancy or by hormonal treatment – is equally effective regardless of whether it takes place before or after exposure to the carcinogen 383948. Importantly, pregnancy-associated protection, when applied after carcinogen exposure, does not rid the gland of the transformed cells during the differentiation and involution processes as evident by the fact that microtumors are present in the mammary gland of parous animals regardless of whether the carcinogen exposure takes place before or after pregnancy occurs 48.

Therefore, tumor formation does take place in the mammary gland of parous rats with frequency almost equal to that in age-matched virgin animals, but only the tumors in the virgin rats progress to palpable size. We do not yet understand the reason for this, but the differences here must be subtle and may only become apparent after the transformation of the mammary tissue. That is, parity would have little effects on growth of normal mammary tissues, but after transformation the environment of the mammary tumors will not stimulate growth of the cancerous cells. It is not an unknown phenomenon that mammary tumors require different environmental factors for growth from those of normal mammary epithelia. For example, some 40 years ago Huggins and coworkers 49 demonstrated that treatment of rats carrying chemically induced mammary cancers with 17β-estradiol and progesterone destroyed over 50% of their tumors, whereas the same hormonal treatment caused an ample development of the normal mammary epithelia. They also pointed out that hormonal deprivation, such as ovariectomy, kills a large percentage of hormone dependent mammary tumors, whereas the normal mammary epithelia remain viable, only exhibiting slower growth and metabolic rates 49. Also, when normal mammary epithelia from virgin rats are transplanted into the cleared fat pad of parous rats, growth of these normal cells is no different from the growth of normal cells transplanted into virgin host. That is, the hormonal environment of the parous animals is just as capable of stimulating growth of normal mammary epithelia as the environment of the virgin hosts. However, when transformed epithelial cells are transplanted into cleared fat pads of virgin and parous hosts, the hormonal environment of the virgin hosts is significantly more favorable for growth of the transformed cells than that of the parous animals 36.

When we administered IGF-I to the parous animals, the environment of the transformed cells was sufficiently altered for them to progress to palpable tumors. The growth promoting activity of IGF-I on mammary cancer cells is well established. For example, when a number of different mitogens were tested for growth promoting activity in MCF-7 and T47D breast cancer cells, IGF-I was shown to have the highest mitogenic activity 50. We found in the present study that although the total expression of ERK1/2 did not differ between the animal groups, the activity (phosphorylation) of ERK1/2 was significantly elevated in IGF-I-treated rats as compared with the other groups. This indicates that activity of the Raf/Ras/mitogen-activated protein kinase (MAPK) cascade was significantly increased in the mammary glands of IGF-I-treated animals. Concomitant with the increased activity of the Raf/Ras/MAPK cascade, we found a significant increase in the expression of the PR in mammary tissues from IGF-I-treated parous rats. Because ERK1/2 phosphorylates (activates) ER-α 51 and because ER-α is an essential regulator of the expression of PR 52, we conclude from these results that the IGF-I treatment caused increased activation of the MAPK cascade, resulting in increased activation of ER-α, which in turn enhanced its transcriptional activity and tumor growth.

Interactions between the IGF-I and ER-α/17β-estradiol in regulating mammary gland development are unequivocal. For example, very limited mammary epithelial development will take place in the total absence of IGF-I, as demonstrated using the IGF-I^-/-mouse model, and administration of 17β-estradiol alone, even in supraphysiological doses, was completely ineffective in restoring mammary epithelial growth in the IGF-I knockout animals 18. Thus, ER-α is practically inactive in the mammary gland in the absence of IGF-I. IGF-I stimulates the expression of 17β-estradiol-regulated genes such as the PR, pS2, and LIV-1 in breast cancer cells, apparently through the ER-α, because this IGF-I activity is obliterated by antiestrogens 5354. Furthermore, the Raf/Ras/MAPK cascade is one of the major cell signaling systems to mediate the effect of IGF-I on ER-α 51. Therefore, we demonstrated in the present study that IGF-I acts very similarly in the mammary gland when administered in vivo as it does when used for cultured breast cancer cells, in that ER-α is activated via the Raf/Ras/MAPK cascade.

Further delineating the similarity between this rat study and studies carried out on cultured breast cancer cells is the recent finding that IGF-I treatment of MCF-7 cells sharply reduces the total expression of the ER-α, while significantly increasing the expression of the PR and pS2 – an activity that is blocked with antiestrogen 5556. Those investigators showed that the phosphatidylinositol 3-kinase (PI3-K)/Act pathway was involved in regulating the enhanced PR expression. However, the effects of the PI3-K/Act pathway on PR expression is complex, because Cue and coworkers 57 found an indication of an IGF-I-induced inhibition of PR expression mediated through the PI3-K/Act cascade – an activity completely independent of ER-α. Therefore, the effects of IGF-I on PR expression may differ depending on whether they are achieved through stimulating ER-α activity or they are mediated independent of ER-α.

How the two major signal transduction system activated by IGF-I receptor/IGF-I, the PI3-K/Act pathway and the Raf/Ras/MAPK cascade interact to coordinate the activity of IGF-I in the mammary epithelia is still largely an unanswered question, but the two signaling pathways appear to have extensive interactions with each other 5859. Also, we do not know whether IGF-I is acting here independent of 17β-estradiol to stimulate ER-α, but results from cell culture studies have shown that IGF-I can activate ER-α in the complete absence of estrogen 535460 as well as acting with 17β-estradiol 5356 to stimulate ER-α. However, we predict that 17β-estradiol is indispensable from the IGF-I activity described here.

The IGF-I stimulated increase in ER-α activity could be of fundamental importance to mammary tumor progression in the parous animals. We know that regulation of ER-α in normal tissues is under tight control; for example, increased ER-α activity by estrogen administration causes a sharp downregulation in ER-α 5261. We have seen that administering low doses of 17β-estradiol to parous rats, while unequivocally causing a significant stimulation of mammary development and a large increase in mammary tumorigenesis, will cause a significant reduction in mRNA levels of ER-α in the normal mammary gland as compared with those in untreated parous rats that are refractory to tumorigenesis and age-matched virgin animals. However, no difference was found in ER-α levels in tumors from 17β-estradiol-treated parous rats and age-matched virgin animals (Thordarson G, McCarty M, unpublished data). That is, the tight regulation of ER-α appears to be diminished in mammary tumors compared with normal mammary tissue in that its ligand-mediated down-regulation is lost or at least substantially blunted. This is supported by our previous study, which showed that ER expression is significantly increased in MNU-induced rat mammary tumors as compared with normal mammary tissue 2, and may resemble what has been described in the human breast where a dissociation is found between ER expression and proliferation of normal mammary epithelia, but this dissociation is lost, or is weaker, in breast cancer 62. We hypothesize that this lax regulation of ER-α in transformed cells is a fundamental step in tumor development, because an increase in ER-α activity, caused by 17β-estradiol or other agents that are capable of activating the ER-α, no longer provides negative feedback on ER-α expression, leading to uncontrolled growth and ultimately tumor formation.

Conclusions

IGF-I treatment increases the level of mammary tumorigenesis in parous rats to that in age-matched virgin animals. Based on expression analyses of genes that are known to regulate mammary gland development, we found no clear indication of differences in the susceptibility of the mammary tissues from parous and age-matched virgin rats at the time of carcinogen exposure. However, we did find strong evidence for an increase in the growth promoting activity of mammary tissues from IGF-I-treated rats as compared with animals not receiving IGF-I treatment, in that the activity of the MAPK cascade was elevated with a concomitant increase in activation of ER-α. Furthermore, we hypothesize that the cancerous mammary epithelial cells sustain a defect in the ligand-dependent downregulation of ER-α, and that this defect in regulation of ER-α causes uncontrolled growth of the transformed cells in a hormonal environment with stimulatory pressure on the ER-α system, but they remain latent in a hormonal environment lacking this ER-α stimulation.

Competing interests

None declared.

Abbreviations

ER = estrogen receptor; ERK1/2 = extracellular signal-regulated kinase-1/2; GH = growth hormone; IGF = insulin-like growth factor; MAPK = mitogen-activated protein kinase; MNU = N-methyl-N-nitrosourea; PI3-K = phosphatidylinositol 3-kinase; PR = progesterone receptor; TEB = terminal end bud; TGF = transforming growth factor.

Acknowledgement

We thank Katharine Van Horn for excellent assistance with palpating the animals, measuring tumors, and collecting tissues. We are also indebted to Dr Kurt E Ebner for the antiserum to rat α-lactalbumin. We are also grateful to the NIDDK Hormone Distribution Program for supplying the radioimmunoassay reagents for measuring prolactin and GH, and Genentech Inc. for providing the human recombinant IGF-I. This study was supported by USPHS Grants CA-71590 and CA-72598, awarded by the National Cancer Institute.

Age at first birth and breast cancer risk MacMahon B Cole P Lin TM Lowe CR Mirra AP Ravnihar B Salber EJ Valaoras VG Yuasa S Bull World Health Organ 1970 43 209 221 5312521 Refractoriness to mammary tumorigenesis in parous rats: is it caused by persistent changes in the hormonal environment or permanent biochemical alterations in the mammary epithelia? Thordarson G Jin E Guzman RC Swanson SM Nandi S Talamantes F Carcinogenesis 1995 16 2847 2853 7586208 Biological and molecular bases of mammary carcinogenesis Russo J Russo IH Lab Invest 1987 57 112 137 3302534 Susceptibility of the mammary gland to carcinogenesis. II. Pregnancy interruption as a risk factor in tumor incidence Russo J Russo IH Am J Pathol 1980 100 497 512 6773421 Differentiation of the mammary gland and susceptibility to carcinogenesis Russo J Tay LK Russo IH Breast Cancer Res Treat 1982 2 5 73 6216933 Formation and removal of 7,12-dimethylbenz[a]anthracene–nucleic acid adducts in rat mammary epithelial cells with different susceptibility to carcinogenesis Tay LK Russo J Carcinogenesis 1981 2 1327 1333 6799218 Role of hormones in mammary cancer initiation and progression Russo IH Russo J J Mammary Gland Biol Neoplasia 1998 3 49 61 10.1023/A:1018770218022 10819504 Persistent parity-induced changes in growth factors, TGF-beta3, and differentiation in the rodent mammary gland D'Cruz CM Moody SE Master SR Hartman JL Keiper EA Imielinski MB Cox JD Wang JY Ha SI Keister BA Chodosh LA Mol Endocrinol 2002 16 2034 2051 10.1210/me.2002-0073 12198241 Persistent changes in gene expression induced by estrogen and progesterone in the rat mammary gland Ginger MR Gonzalez-Rimbau MF Gay JP Rosen JM Mol Endocrinol 2001 15 1993 2009 10.1210/me.15.11.1993 11682629 Plasma prolactin levels and breast cancer: relation to parity, weight and height, and age at first birth Kwa HG Cleton F Bulbrook RD Wang DY Hayward JL Int J Cancer 1981 28 31 34 7309279 Long-term effect of a first pregnancy on the secretion of prolactin Musey VC Collins DC Musey PI Martino-Saltzman D Preedy JRK N Engl J Med 1987 316 229 234 3099198 Parous rats regain high susceptibility to chemically induced mammary cancer after treatment with various mammotropic hormones Thordarson G Van Horn K Guzman RC Nandi S Talamantes F Carcinogenesis 2001 22 1027 1033 10.1093/carcin/22.7.1027 11408345 Induction of mammary carcinoma in hypophysectomized rats treated with 3-methylcholanthrene, oestradiol-17β, progesterone and growth hormone Young S Nature 1961 190 356 357 Rat mammary carcinoma regressions during suppression of serum growth hormone and prolactin Rose DP Gottardis M Noonan JJ Anticancer Res 1983 3 323 326 6139974 Somatostatin analogue octreotide enhances the antineoplastic effects of tamoxifen and ovariectomy on 7,12-dimethylbenz(<it>a</it>)anthracene-induced rat mammary carcinomas Weckbecker G Tolcsvai L Stolz B Pollak M Bruns C Cancer Res 1994 54 6334 6337 7987824 Reduced mammary gland carcinogenesis in transgenic mice expressing a growth hormone antagonist Pollak M Blouin M-J Zhang J-C Kopchick JJ Br J Cancer 2001 85 428 430 10.1054/bjoc.2001.1895 11487276 High frequency of mammary adenocarcinomas in metallothionein promoter-human growth hormone transgenic mice created from two different stains of mice Tornell J Carlsson B Pohjanen P Wennbo H Rymo L Isaksson O J Steroid Biochem Molec Biol 1992 43 237 242 10.1016/0960-0760(92)90213-3 1525063 Insulin-like growth factor I is essential for terminal end bud formation and ductal morphogenesis during mammary development Ruan W Kleinberg DL Endocrinology 1999 140 5075 5081 10.1210/en.140.11.5075 10537134 Involution of the lactating mammary gland is inhibited by the IGF system in a transgenic mouse model Neuenschwander S Schwartz A Wood TL Roberts CT Jr Henninghausen L LeRoith D J Clin Invest 1996 97 2225 2232 8636401 Targeted expression of des(1–3) human insulin-like growth factor I in transgenic mice influences mammary gland development and IGF-binding protein expression Hadsell DL Greenberg NM Fligger JM Baumrucker CR Rosen JM Endocrinology 1996 137 321 330 10.1210/en.137.1.321 8536631 IGF and insulin action in the mammary gland: lessons from transgenic and knockout models Hadsell DL Bonnette SG J Mammary Gland Biol Neoplasia 2000 5 19 30 10.1023/A:1009559014703 10791765 Circulating concentrations of insulin-like growth factor I and risk of breast cancer Hankinson SE Willett WC Colditz GA Hunter DJ Michaud DS Deroo B Rosner B Speizer FE Pollak M Lancet 1998 351 1393 1396 10.1016/S0140-6736(97)10384-1 9593409 Elevated growth hormone levels in sera from breast cancer patients Emerman JT Leahy M Gout PW Bruchovsky N Horm Metab Res 1985 17 421 424 4054832 Plasma insulin-like growth factor-1 (IGF-1) concentrations in human breast cancer Peyrat JP Bonneterre J Hecquet B Vennin P Louchez MM Fournier C Lefebvre J Demaille A Eur J Cancer 1993 29A 492 497 8435198 Hormone/growth factor interactions mediating epithelial/stromal communication in mammary gland development and carcinogenesis Imagawa W Pedchenko VK Helber J Zhang H J Steroid Biochem Molec Biol 2002 80 213 230 10.1016/S0960-0760(01)00188-1 11897505 The effect of GH on estrogen receptor expression in the rat mammary gland Feldman M Ruan W Tappin I Wieczorek R Kleinberg DL J Endocrinol 1999 163 515 522 10588825 Dose-responsive induction of mammary gland carcinomas by the intraperitoneal injection of 1-methyl-1-nitrosourea Thompson HJ Adlakha H Cancer Res 1991 51 3411 3415 2054781 Prevention of mammary carcinogenesis by short-term estrogen and progestin treatments Rajkumar L Guzman RC Yang J Thordarson G Talamantes F Nandi S Breast Cancer Res 2003 6 R31 R37 10.1186/bcr734 14680498 Lactogenic response of cultured mouse mammary epithelial cells to mouse placental lactogen Thordarson G Villalobos R Colosi P Southard J Ogren L Talamantes F J Endocrinol 1986 109 263 274 3711764 Preparing mammary gland whole mounts from mice Rasmussen SB Young LJT Smith GH In Methods in Mammary Gland Biology and Breast Cancer Research New York: Kluwer Ip MM, Asch BB 2000 75 85 Tissue concentrations of somatomedin C: further evidence for multiple sites of synthesis and paracrine or autocrine mechanisms of action D'Ercole AJ Stiles AD Underwood LE Proc Natl Acad Sci USA 1984 81 935 939 6583688 Prolactin receptor expression in the epithelial and stroma of the rat mammary gland Camarillo IG Thordarson G Moffat JG Van Horn KM Binart N Kelly PA Talamantes F J Endocrinol 2001 171 85 95 11572793 Establishing a framework for the functional mammary gland: from endocrinology to morphology Hovey RC Trott JF Vonderhaar BK J Mammary Gland Biol Neoplasia 2002 7 17 38 10.1023/A:1015766322258 12160083 The GH-IGF-I axis and breast cancer Laban C Bustin SA Jenkins PJ Trend Endocrinol Metab 2003 14 28 34 10.1016/S1043-2760(02)00003-6 Growth hormone, IGF-I and cancer. Less intervention to avoid cancer? More intervention to prevent cancer? Holly JMP Gunnell DJ Smith GD J Endocrinol 1999 162 312 330 Changes in the parous rat mammary gland environment are involved in parity-associated protection against mammary carcinogenesis Abrams TJ Guzman RC Swanson SM Thordarson G Talamantes F Nandi S Anticancer Res 1998 18 4115 4122 9891455 Prevention of mammary carcinogenesis in rats by pregnancy: effect of full-term and interrupted pregnancy Sinha DK Pazik JE Dao TL Br J Cancer 1988 57 390 394 3134040 Hormonal prevention of breast cancer: mimicking the protective effect of pregnancy Guzman R Yang J Rajkumar L Thordarson G Chen X Nandi S Proc Natl Acad Sci USA 1999 96 2520 2525 10.1073/pnas.96.5.2520 10051675 Short-term exposure to pregnancy levels of estrogen prevents mammary carcinogenesis Rajkumar L Guzman RC Yang J Thordarson G Talamantes F Nandi S Proc Natl Acad Sci USA 2001 98 11755 11759 10.1073/pnas.201393798 11573010 Short-term exposure to estrogen and progesterone induces partial protection against <it>N</it>-nitroso-<it>N</it>-methylurea-induced mammary tumorigenesis in Wistar–Furth rats Medina D Peterson LE Moraes R Gay J Cancer Lett 2001 169 1 6 10.1016/S0304-3835(01)00507-9 11410318 Molecular mechanism of growth hormone (GH) deficiency in the spontaneous dwarf rat: detection of abnormal splicing of GH messenger ribonucleic acid by the polymerase chain reaction Takeuchi T Suzuki H Sakurai S Nogami H Okuma S Ishikawa H Endocrinology 1990 126 31 38 2152867 The growth hormone-deficient spontaneous dwarf rat is resistant to chemically induced mammary carcinogenesis Swanson SM Unterman TG Carcinogenesis 2002 23 977 982 10.1093/carcin/23.6.977 12082019 Mammary tumorigenesis in growth hormone deficient dwarf rats; effects of hormonal treatments Thordarson G Semaan SJ Low C Ochoa D Leong H Rajkumar L Guzman RC Nandi S Talamantes F Breast Cancer Res Treat Short-term treatment with pregnancy levels of estradiol induces protection from mammary carcinogenesis and results in a persistent reduction in growth hormone and prolactin [abstract] Guzman RC Rajkumar L Thordarson G Yang J Reddy M Laxminarayan S Nandi S 93rd Annual Meeting of the American Association for Cancer Research, 6–10 April 2002; San Francisco. Linthicum, MD: Cadmus Professional Communications 2002 2537 Regulation of cyclin-Cdk activity in mammarlian cells Obaya AJ Sedivy JM Cell Mol Life Sci 2002 59 126 142 10.1007/s00018-002-8410-1 11846025 Mammary hyperplasia and carcinoma in MMTV-cyclin D1 transgenic mice Wang TC Cardiff RD Zukerberg L Lees E Arnold A Schmidt EV Nature 1994 369 669 671 10.1038/369669a0 8208295 TGF-β signaling in mammary gland development and tumorigenesis Wakefield LM Piek E Bottinger EP J Mammary Gland Biol Neoplasia 2001 6 67 82 10.1023/A:1009568532177 11467453 Protective effects of pregnancy and lactation against N-methyl-N-nitrosourea-induced mammary carcinomas in female Lewis rats Yang J Yoshizawa K Nandi S Tsubura A Carcinogenesis 1999 20 623 628 10.1093/carcin/20.4.623 10223190 Extinction of experimental mammary cancer, I. Estradiol-17β and progesterone Huggins C Moon RC Morii S Proc Natl Acad Sci USA 1962 48 379 386 14449778 Differential responsiveness of human breast cancer cell lines MCF-7 and T47D to growth factors and 17beta-estradiol Karey KP Sirbasku DA Cancer Res 1988 48 4083 4092 3289739 Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase Kato S Endoh H Masuhiro Y Kitamoto T Uchiyama S Sasaki H Masushige S Gotoh Y Nishida E Kawashima H Metzger D Chambon P Science 1995 270 1491 1494 7491495 Cellular expression of estrogen and progesterone receptors in mammary glands: regulation by hormones, development and aging Shyamala G Chou Y-C Louie SG Guzman RC Smith GH Nandi S J Steroid Biochem Mol Biol 2002 80 137 148 10.1016/S0960-0760(01)00182-0 11897499 Regulation of progesterone receptor gene expression in MCF-7 breast cancer cells: a comparison of the effects of cyclic adenosine 3',5'-monophosphate, estradiol, insulin-like growth factor-I, and serum factors Cho H Aronica SM Katzenellenbogen BS Endocrinology 1994 134 658 664 10.1210/en.134.2.658 7507831 Interaction between estradiol and growth factors in the regulation of specific gene expression in MCF-7 human breast cancer cells El-Tanani MKK Green CD J Steroid Biochem Mol Biol 1997 60 269 276 10.1016/S0960-0760(96)00226-9 9219917 Role of insulin-like growth factor-I in regulation estrogen receptor-α gene expression Stoica A Saceda M Fakhro A Joyner M Martin MB J Cell Biochem 2000 76 605 614 10.1002/(SICI)1097-4644(20000315)76:4<605::AID-JCB9>3.0.CO;2-T 10653980 A role for Akt in mediating the estrogenic functions of epidermal growth factor and insulin-like growth factor I Martin MB Franke TF Stoica GE Chambon P Katzenellenbogen BS Stoica BA McLemore MS Olivo SE Stoica A Endocrinology 2000 141 4503 4511 10.1210/en.141.12.4503 11108261 Insulin-like growth factor-I inhibits progesterone receptor expression in breast cancer cells via the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway: progesterone receptor as a potential indicator of growth factor activity in breast cancer Cui X Zhang P Deng W Oesterreich S Lu Y Mills GB Lee AV Mol Endocrinol 2003 17 575 588 10.1210/me.2002-0318 12554765 Insulin-like growth factor 1 and oestradiol promote cell proliferation of MCF-7 breast cancer cells: new insights into their synergistic effects Dupont J Le Roith D J Clin Pathol Mol Pathol 2001 54 149 154 10.1136/mp.54.3.149 Interactions between estrogen and insulin-like growth factor signaling pathways in human breast tumor cells Hamelers IHL Steenbergh PH Endocr Rel Cancer 2003 10 331 345 Activation of estrogen receptor-mediated gene transcription by IGF-I in human breast cancer cells Lee AV Weng C-N Jackson JG Yee D J Endocrinol 1997 152 39 47 9014838 Progesterone and estradiol interaction in the regulation of rat uterine weight and estrogen receptor concentration Medlock KL Forrester TM Sheehan DM Proc Soc Exp Biol Med 1994 205 146 153 8108464 Dissociation between steroid receptor expression and cell proliferation in the human breast Clarke RB Howell A Potten CS Anderson E Cancer Res 1997 57 4987 4991 9371488