Xenoestrogens and Genetic Damage in Breast Cancer

Institution: SRI International
Investigator(s): Moire Robertson Creek, Ph.D., MPH -
Award Cycle: 1995 (Cycle I) Grant #: 1KB-0397 Award: $159,053
Award Type: New Investigator Awards
Research Priorities
Etiology and Prevention>Prevention and Risk Reduction: ending the danger of breast cancer

Initial Award Abstract (1995)
Estrogens are a class of hormones critical to female sexual development and functioning, but, paradoxically, strong evidence links them with the induction of breast cancer. Precisely which estrogen is responsible is unknown, but high levels of 16a-hydroxyestrone (16a-OHE), a metabolite of the estrogen estradiol, are strongly correlated with the susceptibility to and presence of breast cancer in human and animals. Additional evidence suggests that certain dietary and environmental factors may alter estrogen production or metabolism and thereby function as "xenoestrogens." Xenoestrogens are broadly defined as substances that alter normal estrogen homeostasis (regulation) and function. For example, dietary substances found in cruciferous vegetables, e.g., broccoli or cauliflower, lower the effective levels of 16a-OHE and decrease mammary tumor incidence by inducing competing metabolic pathways. On the other hand, ethanol is thought to increase tumor formation by enhancing metabolic pathways that form 16a-OHE. Definitive studies showing the relationship between 16a-OHE levels, enzyme induction or inhibition, and xenoestrogen exposure have not been conducted and are the subject of this proposal. These studies will define the relative ability of estradiol and its metabolites (e.g.,16a-OHE) to alter genetic material and will examine how xenoestrogens influence the formation of these genotoxic compounds by altering the metabolic pathways leading to their formation. This knowledge may ultimately lead to effective methods for identifying susceptible populations, detecting early stages of breast cancer, reducing preventable causes of breast cancer, and designing chemicals that will help prevent development of breast cancer. We will address two priority issues of the Breast Cancer ResearchProgram: the role of genotoxic agents in the origin of breast cancer, and early detection of breast cancer.

We hypothesize that 16a-OHE may initiate tumor formation by interacting directly with proteins and DNA, thereby leading to chromosomal breakage or loss. Furthermore, since the metabolism of estradiol to 16a-OHE is dependent upon xenoestrogen-inducible metabolic enzyme systems, we also hypothesize that the formation of this metabolite may be affected by xenoestrogens. Specifically, we will study the chromosome-damaging effects of estradiol and its metabolites, investigate covalent binding of estradiol and its metabolites to large molecules both in cell cultures and in breast tissue of animals with varying susceptibilities to breast cancer, study the effect of selected xenoestrogens on the profile of covalent binding, and examine how xenoestrogens alter estradiol metabolism and estradiol-induced genetic damage.

Final Report (1998)
Cancer cells differ from normal cells in several ways. Cancer cells grow inappropriately, they do not become specialized they should, and they may leave the body site from which they originated, invade the surrounding tissue and grow in other parts of the body. This research will provide critical insights into how cancer cells lose proper control over growth, differentiation and invasion. We have shown that the expression of an intracellular protein, known as Id-l, correlates with the growth state of the cells, and that this protein is down regulated during the gain of function (milk production). In addition, we created murine mammary epithelial cells in which Id-l was always present. We found that these cells were unable to produce milk and, more importantly, the cells expressed an enzyme (gelatinase) and acquired the ability to invade the surrounding microenvironment. By contrast, control cells did not have the above characteristics. We therefore suggested that Id- l could be an important protein whose altered regulation in mammary cells results in a proliferative and invasive potential in breast cancer.

In order to investigate our hypothesis, we examined Id-l expression level in different types of human breast cancer cell lines (a total of nine cell lines has been investigated). Under serum-free culture condition, Id- l protein was found to be constitutively expressed at high levels in the most invasive tumor cell lines (called MDA-MB-23 l, MDA-MB436 and MDA-MB435), and at an intermediate level in another invasive cell line (called Hs578T). Meanwhile, Id-l was expressed at very low levels in less aggressive and non-invasive breast tumor cell lines (called T47D, MCF-7, ZR75-l and SKBR-3). An exception was the invasive BT549 cells which did not express any detectable Id-l. Lake murine epithelial cells which always produce Id-l, MDAMB-23 l, MDA-MB-436, MDA-MB435, and at a lower extent Hs578T cells, also expressed a gelatinase of the same size, whereas the non-invasive T47D, MCF-7, ZR75-1 and SKBR-3 cells did not express it. As sex steroid hormones are known to be important regulators of breast cancer progression and treatment, we also examined the possible connection between the hormones and Id- l expression. We found that estrogen treatment upregulated Id- l expression in T47D cells and that co-treatment of estrogen and progesterone downregulated Id-l expression and inhibited growth in the same cells. More strikingly, we have evidence that Id-l can mediate the effect of the two hormones on the proliferation of breast cancer cells positive for the hormone receptors. When T47D cells were treated with estrogen and Id-l artificially maintained at a low level of expression, cell proliferation was reduced in comparison with treatment of estrogen alone. Meanwhile, T47D cells with constitutive Id-l expression were no longer able to respond to progesterone treatment, and they maintained a relatively high growth rate independent of serum in the culture medium. This might provide important molecular mechanisms for some of previously known hormone actions in treatment of breast cancer, and we propose that Id-l protein is a potential candidate for master regulator of aggressiveness and invasiveness in breast cancer.