Biology of the Normal Breast: The Starting Point
Information regarding the normal breast and its development is surprisingly sparse, in spite of improved methods for detecting smaller breast cancer lesions. It is becoming more imperative to be able to distinguish normal from pre-malignant tissue. We know that the structure of the human breast consists of fatty tissue with ducts running through it. We also know that the normal breast is composed of several different types of cells. The fatty tissue has adipocytes, blood vessels and extracellular matrix that provide the support structure for the ducts. The ducts of the breast are lined with epithelial cells, which are the cells responsible for producing the milk and the cells that give rise to 80% of all breast tumors.
This year the BCRP funded three RFA projects to increase our understanding of the normal breast. Two of the grants investigate the physiological changes that occur during the normal progression of the breast through puberty and pregnancy. Satyabrata Nandi will investigate why the mammary glands in rats that have never had offspring are more susceptible to carcinogens than the glands in rats that have (there is a similar pattern in humans), by comparing the genes that are turned on in each type of gland. Mary Barcellos-Hoff will determine whether a growth factor called TGF-b has a significant role in the development of the breast. TGF-b may be important in this process because 1) it is associated with epithelial cell death, which is a necessary step in the maturation of the gland and 2) it is regulated by estrogen and progesterone at different points of breast development. Understanding the role of TGF-b in breast development could lead to finding new ways to stop tumor epithelial cells from growing. Vito Quaranta will determine whether the processing of a component of the extracellular matrix, Ln-5, has a significant effect on the structural integrity of the breast ducts. Loss of structural integrity is believed to be the first stage in tumor cell metastasis. These studies demonstrate how developing a deeper understanding of the properties of the normal breast can give us insights into how tumors escape their normal constraints.
Basic Breast Biology – Request for Applications (RFAs)
Hormonal Regulation of TGF-b During Mammary Development
Mary H. Barcellos-Hoff, Ph.D.
Lawrence Berkeley National Laboratory- Life Sciences Division
The development of both the normal breast and breast cancers are regulated by ovarian hormones, progesterone and estrogen. However, it has long been postulated that these hormones have their effects by inducing certain growth factors that regulate cell growth rates. We propose that transforming growth factor-b (TGF-b), which has been found in different studies to both promote and inhibit human breast cancer, is differentially regulated by estrogen and progesterone, and in turn that it mediates breast development.
It is already well established that TGF-b can profoundly inhibit epithelial cell proliferation and morphogenesis. However, it is not known when this action occurs in the normal breast or how it is regulated. The activity of TGF-b is restrained by its production in a latent complex. TGF-b needs to be released from this complex in an extracellular process called activation in order to influence cells. Studies in our laboratory showed that certain antibodies recognize TGF-b only upon its release from the latent complex, i.e. following activation. We believe that activation is a primary regulator of TGF-b action and requires further study in mammary glands in order to understand the complex role of TGF-b in breast biology and cancer.
We have used these antibodies to obtain preliminary data that indicate that the level of activation is variable in mouse mammary epithelial cells (the cells that line the milk ducts and give rise to most mammary cancers) and is associated primarily with periods of mammary gland growth. Our preliminary data show that the endbud, a multicellular structure necessary for ductal elongation, contains a population of cells with a lot of active TGF-b, in addition to proliferating cells and cells undergoing programmed cell death. Likewise during pregnancy, mammary cells are highly proliferative but TGF-b is present in a certain subset. Since TGF-b profoundly inhibits mammary epithelial cell proliferation in culture, activation during growth periods appears paradoxical. However, TGF-b can also induce cell death, or apoptosis, and apoptosis often accompanies periods of proliferation, perhaps in order to insure that growth stops at the appropriate moment, or in order to weed out damaged cells. In the proposed studies we will define the role of TGF-b during these physiological states by determining whether the TGF-b positive cells are undergoing proliferation or apoptosis. We will also determine whether TGF-b positive cells also express the receptors for these hormones.
Our hypothesis is that during the two major periods of mammary growth, i.e. puberty and pregnancy, estrogen and progesterone differentially regulate TGF-b activity, which in turn both positively regulates apoptosis and negatively regulates proliferation. In combination with our TGF-b antibodies, we will use two new transgenic mouse models in which TGF-b or progesterone receptor have been compromised in order to define the contribution of TGF-b to hormonal regulation of mammary growth and development.
Pregnancy Genes Associated With a Risk for Breast Cancer
Satyabrata Nandi, Ph.D.
University of California, Berkeley- Cancer Research Laboratory
It has been known for about thirty years that a full-term pregnancy (parity) by the age of 18 can permanently reduce the risk for developing breast cancer. This phenomenon is considered to be the best normal physiological preventive measure against breast cancer. Likewise, pregnancy in rats and mice also results in reduction of susceptibility to chemically-induced breast carcinogenesis. This indicates that the pregnancy-associated reduction in susceptibility to breast cancer is species-independent. Thus, animals can provide an excellent experimental system to study this process. The ultimate goal of these studies is to generate specific information on pregnancy-associated genetic changes related to the risk of developing breast cancer.
Recently, we have made considerable progress in understanding the biological basis for pregnancy-associated reduction in susceptibility to mammary carcinogenesis in rats and mice. We have confirmed that a short-term hormonal treatment procedure using natural ovarian steroids will prevent the chemical-induced mammary cancer development in rats. These ongoing studies also suggest that the elevated blood levels of estrogens and progesterone during pregnancy are likely to be the major cause of pregnancy-associated reduction in susceptibility to mammary cancer in rats. Information is lacking, however, on the specific genetic changes in breast epithelial cells that occur with either hormonal treatment or pregnancy that could account for this protective effect.
Thus, the current proposal focuses on the specific molecular/genetic factors controlling pregnancy-associated reduction in susceptibility to mammary carcinogenesis. We will study differential gene expression between mouse mammary glands which are either ‘cancer susceptible’ (virgin) or ‘cancer non-susceptible’ (parous or hormone treated virgin). We will utilize a molecular biology technique called "differential display" to identify gene activities associated with either virgin or parous mammary glands. At present, we have a candidate gene, RMT1, which meets some of the criteria for such a pregnancy gene. We will study this gene further and seek to expand our studies to discover additional genes using the differential display technology.
Once genes associated with pregnancy are identified and characterized, these are likely to provide information that can be used to develop molecular genetic strategies for the prevention of human breast cancer. Our hope is that this information will prevent breast cancer via a normal physiological process and enhance the quality of life.
Regulation of Breast Epithelial Cell Motility by Proteases
Vito Quaranta, Ph.D.
The Scripps Research Institute
Breast cancer is lethal because cancer cells are able to invade the surrounding tissues. Eventually, these cells grow at distant sites, and the spread of breast cancer throughout the body becomes life threatening. If one could prevent cancer cells from invading, breast cancer would remain confined and could conceivably be cured by surgical removal of the initial cancerous lesions. Our interest is focused on the very early step of cancer cell invasion, its crossing of the basement membrane. In the normal breast gland, the basement membrane keeps the breast epithelial cells neatly organized in functional structures. Importantly, it keeps them separated from the underlying connective or fat tissues. At early stages, even breast cancer cells are effectively segregated from surrounding tissues by the basement membrane.
This is recognized clinically as carcinoma in situ. A critical transition in the development of breast cancer occurs when cancer cells no longer respect the boundary represented by the breast gland basement membrane. Once they invade the underlying tissues, cancer cells have made the first step to spread to the rest of the body. The transition to invading cells is unpredictable in all cancers, but particularly so in breast cancer. Therefore, there are several important questions to be addressed. What motivates cancer cells to cross the basement membrane? What are the mechanisms that make normal cells respect the basement membrane boundary? How do these mechanisms break down in invading breast cancer? An answer to these questions will form a foundation for novel treatment of breast cancer, specifically aimed at blocking cancer cell invasion and spread (metastasis).
With this proposal, we hope to identify one mechanism of breast cancer cell invasion, which may represent a target for drug discovery. We found that a substance called protease MMP2 acts upon a constituent of the basement membrane, called laminin-5 (Ln-5), and changes it into a stimulant for cell invasion. We showed that breast cells like to adhere to Ln-5 and stay put. In contrast, if breast cells come in contact with Ln-5 that has been modified by the protease MMP2, they become highly motile. It is perhaps more than a coincidence that, in tissue specimens from patients, MMP2 is often found concentrated along sites where breast cancer cell invasion occurs. We believe this phenomenon reflects a mechanism whereby MMP2 stimulates cancer cells to invade by activating a function on Ln-5 that makes cells move around. The basic knowledge about the breast obtained from these studies may form the basis for novel approaches for designing drugs that can stop breast cancer cells from spreading, even at advanced stages.
