Biology of the Normal Breast: the Starting Point
Innovative, Developmental and Exploratory Awards- Type II
Postdoctoral Fellowship Awards
A good deal of research has been applied to understanding the biology of breast tumors biology; however, we still know very little about the structure and biochemistry of the normal breast. BCRP has made studying normal breast biology a priority in hopes that we will be able to sort out early steps that occur during tumor formation and critical turning points.
In 1999, BCRP awarded seven grants that study processes occurring in normal cells and normal breast development. Two postdoctoral fellows are searching for as yet unidentified genes involved in normal cell behavior that could prove to be critical sites for mutations in cancer cells. Jarnail Singh is searching for genes that act as intermediates for the Id-1 gene in influencing on cell movement and Hong Zhang is searching for genes responsible for causing cells to stop dividing (senesce). Two investigators are determining whether genes that regulate the growth and physiology of normal cells in species other than human, or tissues other than breast, are functional in the breast. Peter Jackson is studying POP genes, which when mutated cause yeast to make abnormal amounts of DNA. He is determining whether the POP genes are playing a similar role in the breast. If they are, mutations in these genes could be the basis for oncogene amplification (an early step in the development of cancer) in breast tumors. Carmen Hagios is undertaking a postdoctoral fellowship to investigate the role of two embryonic genes, HOXa-1 and HOXb-7, in normal breast. She will determine whether they are factors in breast development and whether deregulation of these genes leads to tumor formation.
Two investigators are defining the role in the normal breast of cellular factors that have already been correlated with tumor formation, with the ultimate goal of determining whether the factors can be exploited for breast cancer treatment detection or prevention. Wen Xie is using transgenic mice in her postdoctoral fellowship to characterize the effect of extra ACTR/AIB1, a mediator of estrogen action, on normal breast development and susceptibility to cancer. Nitric oxide stimulates breast tumor development, but there are several conflicting theories about how it is achieving its effect. Carol MacLeod is using transgenic mouse models to investigate the effect that removing nitric oxide has on the normal breast, the immune system and tumor development.
Finally, Sheldon Miller will study the secretory process of the breast. The basic function of the breast is to produce milk, which involves the transport of water across the epithelial cells and into the breast ducts. This essential process of breast biology is poorly understood. Moreover, there is a condition called gross cystic disease, which involves the abnormal accumulation of water in the breast ducts. Women with this condition have an increased risk of developing breast cancer when compared to the general population. Dr. Miller's investigation will provide insight into this condition.
Innovative, Developmental and Exploratory Awards – Type II
Role of the POP1 Gene in Breast Cancer Genomic Stability
Peter K. Jackson, Ph.D.
Stanford University
The majority of human breast cancers have an increased content of abnormal DNA in each tumor cell. This increased DNA content is a strong predictor of the prognosis of many solid tumors. In part, the increased DNA content may be due to specific amplifications of specific genes. Our working hypothesis is that alterations in factors controlling the accuracy of DNA replication are important for the development of human breast cancer. The mechanisms underlying this form of genomic instability are essentially unknown.
We have identified a mechanism in yeast critical for regulating DNA content. The activation of two yeast genes, POP1 and POP 2, directs the destruction of regulatory proteins that are responsible for limiting DNA replication. Mutations in POP 1 or POP2 cause yeast to accumulate these regulatory proteins and to replicate their DNA too many times. We have identified human and mouse genes similar to the yeast POP genes. We have begun to test whether these genes serve a similar role in mammalian cells. Using genetic techniques, we have evidence that the human POP1 may be important for restraining cancer growth, and may play a role in restricting breast cancer growth.
The purpose of this project is to find genetic evidence that the human POP genes are actually altered in breast tumors. We will then develop tools to allow us to rapidly diagnose changes in the POP genes, the proteins they encode, or the effects they have in tumor cells.
We believe that the POP genes may be critical caretakers of the cellular DNA. Accordingly, mutations in cellular caretakers may promote the alteration of other genes that either activate cancer (oncogenes) or limit cancer (tumor suppressor genes). This loss of genetic stability is a feature in the late stages of tumors and may be critical for the aggressiveness of specific tumors, including breast tumors.
The Role of Nitric Oxide and Arginine in the Normal and Tumorous Breast
Carol L. MacLeod, Ph.D.
University of California, San Diego Cancer Center
The discovery that the simple gas nitric oxide has important and quite remarkable biological properties electrified the scientific community in the late 1980s. This discovery was awarded the Nobel Prize in Physiology and Medicine in 1998 because of the importance of this substance in controlling blood pressure, regulating nervous system functions and in immune responses. However, the effect of this natural bioactive substance on breast development and breast cancer requires further study in a reliable model system because numerous conflicting studies leave unsettled whether nitric oxide stimulates or inhibits tumor growth and spread (metastasis). Our genetic breast cancer model system has already permitted an answer to part of this question: nitric oxide stimulates tumor growth and the number of tumors that form. It-is now important to determine precisely which cells are producing the damaging nitric oxide that causes this unexpected effect. This is important since nitric oxide is made by all the cells in the breast: (1) in special immune cells that migrate into tumors, (2) in the special cells of the breast designed to produce milk (epithelial); those epithelial cells are the ones that cause breast cancers and, (3) in the fatty cells and "scaffolding cells" that surround, support and nourish the breast epithelial cells (the fat pad). To do this, we will transplant young breast epithelial cells that are genetically programmed to later become tumor cells into a fat pad surgically cleared of epithelial cells. These will either have or lack the ability to make nitric oxide. From our experiments, we will determine the cell types that are producing the damaging nitric oxide.
Our lab also found that when a dietary component of protein (arginine, that is needed for the synthesis of nitric oxide) is removed from the diet of mice, the rate of breast tumor growth and metastasis is significantly reduced. For this reason, studies are proposed to assess the consequences of genetically removing a cellular gateway that permits arginine to enter cells. Without this gateway, we recently discovered immune cells are unable to make nitric oxide. If those cells are important sources of nitric oxide that cause the rapid growth of the breast cancer cells and permit their escape to spread to other body sites, then blocking that gateway might reduce tumor growth and metastasis. Hence, the second aim of our study is to determine if removing that gateway for arginine reduces the rate of breast cancer growth and metastasis.
The results of our studies will provide important leads regarding diet changes that might be beneficial to women and what substances might inhibit nitric oxide. Our findings will prepare the foundation for a further study of women who are at risk for breast cancer relapse. The breast cancer mouse model provides a genetic approach to test directly and conclusively the function arginine transport on nitric oxide formation in breast cancer development and metastasis. It will also determine if the gateway for arginine entry into cells is a good target for the development of new therapeutics. In fact there are several practical reasons to elucidate the specific role of NO in breast cancer. Dietary restriction of L-arginine is known to reduce circulating arginine levels and can improve clinical outcome in a variety of conditions. The identification of the precise arginine gateway involved in NO production may provide new opportunities for therapeutic intervention.
Postdoctoral Fellowship Awards
Hox Genes in Normal Breast Development and Breast Cancer
Carmen Hagios, Ph.D.
Lawrence Berkeley National Laboratory; Life Sciences Division
Cancer is intimately related to the process of development and growth. Every gene implicated in progression to a cancerous phenotype has at least one role, and often multiple roles, during the course of normal growth and development of an embryo. Here, we propose to study Hox genes in the mammary gland because they play important roles in normal breast development, and may have implications in breast cancer.
The mammary gland in many respects is an embryonic organ because it undergoes its major morphological and functional maturation (differentiation) after birth. Dr. Bissell's laboratory and others have found that a set of important developmental genes, known as Hox genes, are present in normal breast cells and furthermore, breast tumors have Hox genes that are not found in the normal breast. We will concentrate on two Hox genes, Hoxa-1 and Hoxb-7, both of which are found in breast epithelial cells cultured on tissue culture plastic outside the animal's body. Studies in the breast in animals have shown that Hoxb-7 is present throughout mammary gland development except during lactation. However, Hoxa-1 was found only in breast tumors. Why is Hoxa-1 present in normal breast cells in culture but absent from the normal breast?
Breast epithelial cells in the body "sit" on a thin layer of a specialized network of globular proteins referred to as extracellular matrix. This protein network provides important positional and practical information that is required for mammary epithelial cell function, including milk production and secretion. Breast epithelial cells in culture are not functional unless in contact with an extracellular matrix that mimics their environment in the body. Under these conditions, we found no Hoxb-7 and Hoxa-1 in normal mammary epithelial cells. We therefore believe that extracellular matrix regulates these genes in the body. Hoxa-1 continues to be present in tumor cells, however, even in the presence of extracellular matrix, indicating that this regulatory mechanism is disrupted in tumor cells.
Based on the presence of Hoxb-7 during stages of branching, and on the absence of Hoxb-7 during lactation in the breast, I propose to explore whether Hoxb-7 is involved in morphogenesis of the breast when the animal is not lactating, and whether this gene must be suppressed so the animal can lactate.
Although the presence of Hoxa-1 and breast cancer strongly coincide, the exact role of Hoxa-1 in breast tumor formation and progression has not yet been addressed. Here I propose to use a genetic engineering approach to increase Hoxa-1 in normal breast cells in order to investigate the effect of a deregulated Hoxa-1 gene on normal breast development in cell culture assays and inside the body. Furthermore, using other techniques, Hoxa-1 expression will be inhibited in tumor cells. Both these engineered cells will then be tested for their potential to grow and to invade in the mouse breast.
Hox genes have been shown previously to have tumorigenic potential when increased in fibroblasts and thus, are often defined as 'cancer genes'. Indeed, if abnormally present, they have the potential to misregulate a large number of genes, and thus, to contribute to cancer pathology. It is therefore conceivable that Hox genes might be targets of future gene therapies in the prevention and treatment of cancer.
Identification of Novel Id-1 Regulated Genes in Breast Cells
Jarnail Singh, Ph.D.
California Pacific Medical Center Research Institute
Breast cancer occurs due to alterations in various mechanisms that control normal cell behavior. Major differences between normal and cancer cells are: a) cancer cells undergo uncontrolled proliferation; b) they generally lose their ability to serve the functions they normally have in a given tissue; c) they gain the ability to migrate and invade other tissues. These aspects are intimately related in breast epithelial cells, which represent the most common (about 90%) source of breast cancer. Normally, these cells should stop growth and invasion when they become functional breast cells, i.e., milk producing cells. Complicated events are required for the normal cells to change from one phenotype to the other. These events involve synthesis and accumulation of certain proteins required for transition from dividing cells to milk producing cells (called the differentiated state). Cancer cells do not synthesize these differentiation-specific proteins. Rather, they keep on making some other proteins that help them to grow continuously, migrate, and invade other tissues. The proposed work in this application is aimed at identifying and characterizing some of the proteins that regulate the ability of normal cells to change from one phenotype to the other, i.e. growing mammary epithelial cells to milk producing cells or cancerous cells.
We have established a cell culture system in which breast epithelial cells can be manipulated either to grow or to become milk protein producing cells. Using this system, it has been found that one protein, named Id-1, regulates the production and activation of other proteins important for normal cell growth or functioning of the breast cells. Its name 'Id' stems from the fact that it inhibits the differentiation of breast cells into milk producing cells. In fact, Id-1 helps the breast epithelial cells continue to grow, which is one of the characteristics of cancer cells. In addition, investigations in our lab have also shown that Id-1 triggers the cells to migrate and spread to other tissues of the body. This spreading of cancer cells is, in reality, the major cause of death in patients with breast cancer.
Since the profound effects of Id-1 on cell phenotype are mediated mainly through the regulation of other genes (proteins), we will try to identify these downstream target genes important for the normal growth, development, and function of breast cells. The proposed project, therefore, would provide vital clues into the intricate mechanisms of breast cell development and the reasons for the uncontrolled proliferation and invasion in cancerous cells. It has the potential of proving to be helpful in the early diagnosis and treatment of breast cancers.
Cloning of Senescence Genes in Mammary Epithelial Cells
Hong Zhang, Ph.D.
Stanford University School of Medicine
Cancer is a group of diseases resulting from changes in genes that control cell growth and behavior. To become malignantly transformed, a normal human cell needs to acquire genetic changes in multiple critical genes. Alterations in many genes that control cell growth and death can contribute to neoplastic transformation. Normal primary human cells have limited life spans, and they can only proliferate for a limited number of time before entering a stage called cellular senescence. One of the most common changes in cancer cells is that they have lost the ability to undergo senescence and can divide indefinitely becoming "immortal". Understanding of the genetic basis for senescence in cells of the human breast will enable the development of new therapeutic approaches and new management measures for cancers. However, the genetic basis for cellular senescence is not well understood.
The overall goal of the proposed study is to identify the genes that control cellular senescence in human mammary epithelial cells, and study the functions of isolated senescence genes. The proposed study will employ a new genetic procedure that was recently developed in my mentor's laboratory. This procedure allows the identification of previously unknown genes that play a crucial role in the control of cellular senescence. Identification of genes and genetic pathways of cellular senescence will provide therapeutic targets for restoration of cellular senescence in breast cancer cells. Other translational potential of the proposed work includes the design of epidemiological approaches for accessing the genetic susceptibility of individuals to develop breast cancer and the creation of prognostic tests for managing breast cancer.
ACTR/A1B1 in Mammary Gland Development and Tumorigenesis
Wen Xie, Ph.D.
The Salk Institute
Most breast cancer arises from the uncontrolled growth of mammary epithelial cells. The steroid hormone, estrogen, stimulates the growth of mammary epithelial cells. Estrogen is required for the normal growth of the breast and is a prerequisite for breast cancer formation. Blocking of estrogen receptor or estrogen action results in tumor regression in breast cancer patients with hormone-dependent tumors. Recently, a group of proteins called "nuclear receptor co-factors" has been identified that play a central role in regulating the function of steroid hormones and their receptors.
ACTR/AIB1 (Amplified in Breast Cancer-1), a nuclear receptor coactivator, interacts with the estrogen receptor and enhances the biologic effects of estrogen. In addition, overproduction of ACTR/AIB1 has been found in breast cancers and in human breast cancer cells lines. ACTR/AIB1's ability to enhance the function of estrogen receptor in addition to its overproduction in breast cancer implicate ACTR/AIB1 as an important player in estrogen response and as a potential contributor to the initiation and progression of breast cancer. In order to determine the significance of ACTR/AIB1 in breast cancer, it is necessary to investigate the biologic effects of ACTR/AIB1 in animal models.
To examine the role that ACTR/AIB1 plays in the control of breast development and breast cancer formation, we will generate transgenic mice in which ACTR/AIB1 is particularly overproduced in the breast. We will perform experiments to examine: 1) the changes in the development of the breast as the result of overproduction of ACTR/AIB1; 2) whether the overproduction of ACTR/AIB1 is sufficient to cause breast cancer; and 3) whether ACTR/AIB1 can accelerate breast cancer formation by other known cancer-causing agents. The results of these experiments will enhance our understanding of normal breast development and of breast cancer pathogenesis. Furthermore, if ACTR/AIB1 is proved to promote the formation of breast cancer, blocking ACTR/AIB1 signaling might be a useful strategy in developing novel therapies for breast cancer.
