Based on our studies of Sprague Dawley rat mammary carcinogenesis induced by environmental polycyclic aromatic hydrocarbons, we have determined that the mammary gland susceptibility to carcinogenesis is closely dependent of the presence of highly proliferating terminal end buds (TEBs) i. These structures are prevalent during a specific “window” of sexual development encompassed between the initiation of ovarian function and the first pregnancy. During this “window”, exposure to different factors such as endocrine disruptors or hormonal treatments that could possibly alter the circadian master clock and affect peripheral clocks in the mammary gland could play an important role in breast cancer initiation and progression.
Epidemiological observations have demonstrated that exposure to radiation at a young age , ii, iii and smoking at puberty , iv, v increase the lifetime breast cancer risk in women. These findings confirm experimental observations that the susceptibility of the breast epithelium to be transformed by a carcinogen is maximal while traversing the high susceptibility window of puberty .vi Therefore the possibility exists that women would be at higher risk of cancer initiation during specific stages of breast development, especially during puberty and before a full term pregnancy. During this period the mammary gland parenchyma undergoes marked longitudinal growth and branching driven by the TEBs, which are composed of a multilayered and actively proliferating epithelium that give origin to ductal adenocarcinomas. Cancer initiation is profoundly inhibited in rats that had completed one pregnancy or had received a 21-day treatment with the placental hormone chorionic gonadotropin (hCG) prior to DMBA administration .vii During the first pregnancy the immature breast undergoes rapid growth with rapid cell division under the influence of hormones secreted by the embryo, mainly hCG, the ovarian hormones estrogen and progesterone, and the pituitary hormone prolactin .viii As the pregnancy progresses the hormonal influences induce the development of lobules and milk secretion, resulting in permanent genomic changes in the breast epithelium that result in inhibition of cancer initiation .ix It is possible that environmental exposures alter the sequence of growth to differentiation, thus inhibiting the activation of genes that are needed for the protection of the breast from cancer development .x
Among environmental exposures, light plays an important role because it synchronizes the circadian rhythm and could act as an endocrine disruptor that could influence the incidence of breast cancer, such as the increase reported in women doing shift work, including flight attendants and women working at night. A reduction in breast cancer incidence in blind women further confirms the role of light exposure on breast cancer risk. All biological processes exhibit rhythmic oscillations every 24 hours that characterize the circadian rhythms. These rhythms are centrally controlled by the master circadian clock located in the suprachiasmatic nucleus (SCN) in the anterior hypothalamus. It is in turn daily synchronized by light input from the retina. Circadian rhythms and cell division are fundamental biological systems in most organisms. There is substantial evidence that, in mammals, circadian rhythms affect the timing of cell divisions in vivo. The development and differentiation of the mammary gland, are regulated by pituitary and ovarian hormones that are, in turn, under the control of SCN. Molecular clocks control cell-proliferation rhythms by regulating the expression of cell-cycle genes. The development and differentiation of the mammary gland are regulated in a time-dependent manner that determine the susceptibility of this organ to be transformed by environmental factors, like changes in the light patterns, which will ultimately alter the circadian rhythm and create a time of greater susceptibility to carcinogens (Figure 25: Click to see).
Our studies have been designed with the objectives of characterizing the endogenous pattern of expression of clock genes in the mammary gland of virgin Sprague-Dawley rats in order to determine whether this pattern is varies along the different stages of mammary gland development under standard light conditions. In order to study the expression of circadian rhythm related genes, four groups of Sprague Dawley rats were maintained under standard light conditions (12 light:12 hr darkness) for 2 weeks for collection of mammary tissues at the ages of 21, 35, 50 and 100 days (Figure 26: Click to see).
Four rats from each group were euthanized every four hours, at Zeitgeber times (ZT): ZT2, ZT6, ZT10, ZT14, ZT18, ZT22 (ZT0 -lights on at 6:00 hr and ZT12 — lights off at 18:00 hr — Figure 27: Click to see).
Mammary glands were dissected for RNA extraction; Real Time RT-PCR analysis was performed for the study of the following clock genes: Period (Per)1, 2, and 3, Cryptochrome (Cry)1 and 2, basic helix-loop (bhlhb)2 and 3, brain and muscle ARNT-like protein 1 (Bmal1) and Clock. Each gene was normalized (ΔCT) using β-actin as an endogenous control gene. Analysis with two-way analysis of variance (ANOVA) showed that the mean expression level for all the genes was significantly different across age and time point as well the interaction between them, with a false discovery rate less than 1%. Since all of the interactions were found to be significant a one-way ANOVA test was performed separately for age and time. The results obtained from the one-way ANOVA by time reveled that in the age groups 21 and 35, eight of the nine genes studied showed significant differences between the six time points observed under this study. The one-way ANOVA by age group indicated a main effect of mammary gland developmental stages on all the genes studied at certain time points. The 35 day group showed significant gene expression changes when compared with the other age groups, such as consistent lower level of expression at ZT6 and ZT10 in Per1, Per2, BHLHB2, and Clock. Of great interest was our finding that the expression of Bmal1 in the 35 day group exhibited a significant higher level of expression at ZT14 and ZT18 when compared to the other age groups.
Our results confirm that the rat mammary gland expresses clock genes that cycle in response to circadian rhythms. Furthermore, these results support the novel finding that clock genes are more greatly modified at the time of vaginal opening (puberty). Since the differentiation of the mammary gland is regulated by the SCN, changes in the circadian rhythm could result in deregulation of basic developmental processes and greater susceptibility to carcinogenesis.
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|vii||Russo IH, Koszalka M, Russo J: Human chorionic gonadotropin and rat mammary cancer prevention. J Natl Cancer Inst 82: 1286-1289, 1990.|
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|ix||Russo J, Balogh GA, Heulings R, Mailo DA, Moral R, Russo PA, Sheriff F, Vanegas J, Russo IH. Molecular basis of pregnancy-induced breast cancer protection. Eur J Cancer Prev. 15(4):306-342, 2006.|
|x||Russo, J., Balogh, G.A., Russo, I.H., and the FCCCHospital Network Participants. Full term pregnancy induces a specific genomic signature in the human breast. Cancer Epidemiol. Biomarkers and Prevention, 17(1): 51-66, 2008.|