Development of mammary cancer in γ-irradiated F1 hybrids of susceptible Sprague-Dawley and resistant Copenhagen rats, with copy-number losses that pinpoint potential tumor suppressors.

Copenhagen rats are highly resistant to mammary carcinogenesis, even after treatment with chemical carcinogens and hormones; most studies indicate that this is a dominant genetic trait. To test whether this trait is also dominant after radiation exposure, we characterized the susceptibility of irradiated Copenhagen rats to mammary carcinogenesis, as well as its inheritance, and identified tumor-suppressor genes that, when inactivated or mutated, may contribute to carcinogenesis. To this end, mammary cancer-susceptible Sprague-Dawley rats, resistant Copenhagen rats, and their F1 hybrids were irradiated with 4 Gy of γ-rays, and tumor development was monitored. Copy-number variations and allelic imbalances of genomic DNA were studied using microarrays and PCR analysis of polymorphic markers. Gene expression was assessed by quantitative PCR in normal tissues and induced mammary cancers of F1 rats. Irradiated Copenhagen rats exhibited a very low incidence of mammary cancer. Unexpectedly, this resistance trait did not show dominant inheritance in F1 rats; rather, they exhibited intermediate susceptibility levels (i.e., between those of their parent strains). The susceptibility of irradiated F1 rats to the development of benign mammary tumors (i.e., fibroadenoma and adenoma) was also intermediate. Copy-number losses were frequently observed in chromosome regions 1q52-54 (24%), 2q12-15 (33%), and 3q31-42 (24%), as were focal (38%) and whole (29%) losses of chromosome 5. Some of these chromosomal regions exhibited allelic imbalances. Many cancer-related genes within these regions were downregulated in mammary tumors as compared with normal mammary tissue. Some of the chromosomal losses identified have not been reported previously in chemically induced models, implying a novel mechanism inherent to the irradiated model. Based on these findings, Sprague-Dawley × Copenhagen F1 rats offer a useful model for exploring genes responsible for radiation-induced mammary cancer, which apparently are mainly located in specific regions of chromosomes 1, 2, 3 and 5.

Differential effect of parity on rat mammary carcinogenesis after pre- or post-pubertal exposure to radiation.

Radiation exposure during the peri-pubertal period is a proven risk factor for breast cancer, whereas parity is an established protective factor. The present study investigated whether parity imposes differential protective effects against radiation-induced rat mammary carcinoma depending on the age at exposure. Pre- and post-pubertal female rats, irradiated or left unirradiated, were mated and allowed to nurse until weaning or left unmated. Appearance of mammary tumors was monitored, and serum concentrations of estradiol and progesterone were measured following weaning. Carcinomas were evaluated by immunohistochemistry for estrogen receptor, progesterone receptor, and the cell proliferation marker Ki-67. Parity reduced the risk of carcinoma in unirradiated and pre-pubertally irradiated rats but not post-pubertally irradiated rats. Although radiation exposure increased serum progesterone level, parity after pre-pubertal exposure significantly decreased the elevated progesterone to a normal level, reflecting a protective effect. Moreover, parity significantly decreased the proportion of hormone receptor-positive carcinomas after pre-pubertal exposure. Parity was also related to the observed positive association between progesterone receptor and Ki-67 indices in cancer tissue, implying progesterone receptor-dependent cell proliferation. Thus, parity protects against radiation-induced rat mammary carcinogenesis depending on the age at exposure; the mechanisms may involve changes in hormone levels and cancer tissue.

Age Modifies the Effect of 2-MeV Fast Neutrons on Rat Mammary Carcinogenesis.

The relative biological effectiveness (RBE) of neutrons depends on their physical nature (e.g., energy) and the biological context (e.g., end points, materials). From the perspective of radiological protection, age is an important biological context that influences radiation-related cancer risk, but very few studies have addressed its potential impact on neutron effects. We therefore investigated the influence of age on the effect of accelerator-generated fast neutrons (mean energy, ∼2 MeV) in an animal model of breast carcinogenesis. Female Sprague-Dawley rats at 1, 3 and 7 weeks of age were irradiated with fast neutrons at absorbed doses of 0.0485-0.97 Gy. All animals were kept under specific pathogen-free conditions and screened weekly for mammary tumors by palpation until they were 90 weeks old. Tumors were diagnosed based on histology. Mathematical modeling was used to analyze mammary cancer incidence, collectively using data from this study and a previously reported experiment on 137Cs gamma rays. The results indicate that neutron irradiation elevated the risk of palpable mammary carcinoma with a linear dose response, the slope of which depended on age at time of irradiation. The RBE of neutron radiation was 7.5 ± 3.4, 9.3 ± 3.5 and 26.1 ± 8.9 (mean ± SE) for animals exposed at 1, 3 and 7 weeks of age, respectively. Our results indicate that age of the animal is an important factor influencing the effect of fast neutrons on breast cancer risk.

Analysis of genes involved in the PI3K/Akt pathway in radiation- and MNU-induced rat mammary carcinomas.

The PI3K/AKT pathway is one of the most important signaling networks in human breast cancer, and since it was potentially implicated in our preliminary investigations of radiation-induced rat mammary carcinomas, our aim here was to verify its role. We included mammary carcinomas induced by the chemical carcinogen 1-methyl-1-nitrosourea to determine whether any changes were radiation-specific. Most carcinomas from both groups showed activation of the PI3K/AKT pathway, but phosphorylation of AKT1 was often heterogeneous and only present in a minority of carcinoma cells. The negative pathway regulator Inpp4b was significantly downregulated in both groups, compared with in normal mammary tissue, and radiation-induced carcinomas also showed a significant decrease in Pten expression, while the chemically induced carcinomas showed a decrease in Pik3r1 and Pdk1. Significant upregulation of the positive regulators Erbb2 and Pik3ca was observed only in chemically induced carcinomas. However, no genes showed clear correlations with AKT phosphorylation levels, except in individual carcinomas. Only rare carcinomas showed mutations in PI3K/AKT pathway genes, yet these carcinomas did not exhibit stronger AKT phosphorylation. Thus, while AKT phosphorylation is a common feature of rat mammary carcinomas induced by radiation or a canonical chemical carcinogen, the mutation of key genes in the pathways or permanent changes to gene expression of particular signaling proteins do not explain the pathway activation in the advanced cancers. Although AKT signaling likely facilitates cancer development and growth in rat mammary carcinomas, it is unlikely that permanent disruption of the PI3K/AKT pathway genes is a major causal event in radiation carcinogenesis.