Growth and characterization of N-methyl-N-nitrosourea-induced mammary tumors in intact and ovariectomized rats

Abstract

It is well established that 85-90% of chemically induced mammary tumors in rats will disappear or diminish significantly in size after the ovaries are removed from the animal. However, it is less well established whether a high percentage of these mammary tumors will grow back with prolonged time after ovariectomy. It is also not known what changes in gene expression take place in the tumors as they develop an independence from hormones for growth. This study was carried out to investigate this. Virgin, 50-day-old female Sprague-Dawley rats were injected with N-methyl-N-nitrosourea (MNU) at the dose of 50 mg MNU/kg body wt. When at least one mammary tumor had grown to 1.0-1.5 cm in one dimension, the animal was bilaterally ovariectomized and reduction and then re-growth of the tumors monitored. Control animals were treated identically except they were not ovariectomized when tumors appeared. Re-growths and new tumors and tumors that developed in the control rats were removed when they reached 1.0-1.5 cm in diameter and all animals were killed 25 weeks after the MNU injection. All the animals in the study (100%) developed mammary tumors after MNU injection with an average latency of 56.5 days. After ovariectomy, 93% of the tumors showed 50% or more reduction in size and 76% of the tumors could not be detected by palpation. However, in 96% of the animals where tumor reduction or disappearance occurred, a re-growth or new mammary tumor development took place with an average latency period of 52.8 days from the day of ovariectomy. Of these post-ovariectomy tumors, 36% occurred at a location where tumors had developed prior to ovariectomy, but 64% appeared at new locations. The circulating levels of 17beta-estradiol (E2) was undetectable in the ovariectomized (OVX) rats and significant reduction was seen in the serum concentrations of progesterone (P4), prolactin (PRL), growth hormone (GH) and insulin-like growth factor-I (IGF-I). The tumors from the OVX rats showed indications of progression as evident from loss of differentiation and invasive characteristics. Comparison between tumors from OVX and intact rats revealed a significantly increased expression of P450 aromatase and elevated activation of extracellular signal-regulated kinase 1 and 2, but reduced levels of the progesterone receptor and cyclin D1 in OVX rats. However, the estrogen receptor (ER) content remained similar in tumors from both groups, at least at the protein level, and so did the expression of IGF-I, IGF-II, insulin receptor substrate-1 (IRS1), IRS-2 and epidermal growth factor receptor. IGF-I receptor (IGF-IR) and ErbB-2 were expressed, respectively, in 50 and 70% of the tumors from the OVX animals, whereas these genes were expressed in 100% of the tumors from the intact rats. It is concluded that chemically induced rat mammary tumors may still depend on the ER and local syntheses of E2 and growth factors for growth initially after ovariectomy. However, as these tumors progress, they develop a more aggressive phenotype and lose their dependency on the ER and possibly growth factors.