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-alpha activation via the mitogen-activated protein kinase pathway

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 17beta-estradiol and progesterone. The 17beta-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 17beta-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 alpha-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-alpha 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 D1 and transforming growth factor-beta3 in 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-alpha via the Raf/Ras/mitogen-activated protein kinase cascade.

Figure 1

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.

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.

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 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).

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.

Figure 10

The expression of cyclin D₁ in 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 D₁ antibody (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)-β₃ 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 (100 μg/lane) were electrophorized on a 20% SDS-PAGE; Western blot analysis was carried out using a specific anti-TGF-β₃ antibody (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.