Pathogenesis: Understanding the Disease
Innovative, Exploratory and Developmental Awards –
Type I
Innovative, Exploratory and Developmental Awards –
Type II
New Investigator Awards
Postdoctoral Fellowship Awards
The Pathogenesis BCRP priority issue addresses the topic of breast tumor cell biology. An increased understanding of the initial development and progression of breast cancer at the molecular level lays the groundwork for new approaches and therapies to treat the disease. In 1999, the BCRP funded 19 new grants focused on breast cancer Pathogenesis. These include 1- and 2-year IDEA (Innovative, Developmental, and Exploratory Awards) projects that allow both established investigators and investigators starting out in breast cancer to initiate novel research projects. In addition, Pathogenesis attracted those interested in career development, both Postdoctoral Fellows and New Investigators. This research generally employs the modern tools of molecular biology to understand the unique genes and protein interactions that allow breast cancer to grow, progress, and spread in the body. This is referred to as 'basic science,' since the approach to the disease is at a very fundamental level. This allows breast cancer researchers to integrate information from other cancer types, general cell biology, animal and lower organism model systems, and immunology to applications specific to breast cancer research.
The adhesion, migration, and spread of breast cancer cells in the body continue to be areas of intense research interest. Many of these projects interface with the process of angiogenesis, the development of the tumor blood supply necessary for growth and metastasis. A major clinical advantage to targeting angiogenesis is the ability to attack secondary sites of tumor growth. Jan Schnitzer is funded to investigate the unique surface proteins of the endothelial cells that line blood vessels located within the tumor. Differences between these proteins and normal vascular endothelial cells may provide either direct sites for therapy or the basis for delivery of drugs to breast tumors. Kristiina Vuori is studying a possible mechanism of action of a new anti-angiogenic drug, endostatin, which is believed to interact with an integrin cell adhesion receptor. The major endothelial and breast tumor integrin, avb3, is the subject of two projects. First, an endothelial proteinase will be studied for its binding to this receptor by YingQing Sun. This interaction would serve to facilitate endothelial migration during angiogenesis, and could be the basis for new therapeutics, if confirmed. Secondly, Alex Strongin is studying this same MMP-integrin interaction in breast cancer cells in order to explain how cancer cells are capable of invasion and to explore new approaches to block this movement. Brett Premack will survey breast cancer cells for the presence of chemokines and chemokine receptors. These receptors are important for leukocyte (white blood cell) motility and invasion and, if found on breast cancer, could be new avenues for therapy. Breast cancer cells travel to distant sites via the blood and lymph systems. Brunhilde Felding-Habermann will study the attachment of breast cancer cells to the endothelium by the combined function of the integrin avb3 on the cancer cells and another receptor, ICAM-1, on the endothelial cell. A key aspect of this research is to isolate and study circulating breast cancer cells from the blood of experimental animals. Finally, Sanford Barsky has a unique model system in mice to study inflammatory breast carcinoma, which spreads locally in the breast via the lymph.
He will investigate how this rare form of breast cancer differs from more common types of the disease. Steady progress is being made in explaining how breast cancer cells multiply in an uncontrolled manner. Research under this topic focuses both on the intracellular regulatory systems linked to growth factors and how breast cells evade signals that cause cell death (apoptosis) for normal cells. Elena Pasquale is funded to study a breast cancer receptor signaling kinase for its ability to contribute to growth, metastasis, and tumor progression. The oncogene growth receptor Her-2 is present in about 30% of cancers, and is associated with a poor prognosis. Although the new therapeutic Herceptin was released last year, this topic is of continued research interest. Janis Jackson will investigate how Her-2 and related growth receptors signal through various signaling kinases, the Ras oncogene family. Ronald Weigel will study a gene regulation transcription factor for its role in mediating the estrogen response and as an independent indicator of potential response to therapy. New aspirin-like drugs offer interesting new ways to treat cancer. Youngsoo Kim will focus on the role of COX-2, a target of aspirin-like drugs, for its presence in breast cancer and association with apoptosis pathways. Yang Xu will investigate the major tumor suppressor, p53, for mutations in the regulatory phosphorylation sites as a possible loss or change in function in breast cancer. Finally, Heimo Strohmaier is funded to study the degradation of a cell cycle regulation protein. A failure to regulate the amount of this protein in breast cancer could lead to excess cell growth by permitting cells to pass through cell cycle checkpoints in an uncontrolled manner.
It is well accepted that genetic differences, both subtle
and dramatic, underlie the initiation and progression of breast cancer.
Two projects examine genes involved in breast cancer growth. First, Devon
Thompson has found a new gene, called hAG-2, that appears to associate with
the estrogen receptor and could underlie resistance to anti-estrogen therapy.
Secondly, Katherine Ely will conduct structural analysis of an apoptosis-associated
gene, BAG-1, for its indirect association with the estrogen receptor, which
also could be involved in anti-estrogen resistance. Genetic repair and recombination
within and between different chromosomes lead to severe genetic defects
that characterize advanced forms of breast cancer. Joanna Albala has discovered
and will characterize a new recombination gene, RAD51B, which appears to
be capable of associating with the hereditary breast cancer genes, BRCA1
and BRCA2. DNA damage from environmental causes, such as radiation and chemical
mutagens, must be repaired correctly for cells to remain normal. Eric Brown
will study a potential DNA damage repair gene, called ATR, for its role
in breast cancer and its association with BRCA1 and the tumor suppressor,
p53. Progression of breast cancer involves processes of differentiation,
which are permanent cell changes in genotype and phenotype. Bogi Andersen
will investigate a gene regulation factor, LMO-4, which is involved in white
blood cell differentiation and is thought to be associated with breast cancer
progression. Finally, Pierre-Yves Desprez is funded to continue his studies
on a novel gene regulation factor, called Id-1, which appears essential
for the expression of cell invasion proteases for breast cancer cell migration.
Innovative, Developmental and Exploratory Awards – Type I
Id–1 Expression During Breast Cancer Progression
Pierre-Yves Desprez, Ph.D.
California Pacific Medical Center Research Institute
Breast cancer cells often penetrate the surrounding tissue and eventually metastasize to other body sites. Metastasis is the hallmark of aggressive cancer and normally indicates poor prognosis. Recently, we have found that a specific protein, called Id-1, is associated with mammary epithelial cell phenotypes and accounts for some of the differences between normal and cancerous breast cells in culture. Id-1 is a regulator of a group of gene expression proteins, called transcription factors. The goal of this proposed research is to provide insights into how some of the metastases arise by evaluating the expression of Id-1 in breast cancer biopsies.
We have shown that Id-1 is capable of regulating various aspects of the normal breast function, and we have found that Id-1 protein level has to be tightly regulated in order for the breast cells to properly respond to the signal for growth or gain of milk-producing functions. When such regulation is altered by artificially maintaining a high level of Id-1, cells can no longer respond to the signal for milk production. Additionally, these cells now acquire the ability to break away from each other and dissolve big molecules in their surrounding environment, an invasive property characteristic of aggressive breast cancer cells. To dissolve big molecules, certain enzymes are required. We have identified a novel enzyme, which we call the 120 kD gelatinase, whose production is tightly associated with Id-1 protein, and shown it may be responsible for the ability of breast cells to become invasive. In this project we plan to expand our studies to breast tumor samples using both microscopy and molecular biology techniques to detect and measure the amount of both Id-1 and the 120 kD gelatinase, from biopsy and urine respectively, from the patients.
Since breast cancer is a very heterogeneous disease, biologically and clinically, we hope to find a new marker that indicates the progression of some breast cancers on their way towards their final, untreatable stages. This research will also help to provide a valuable target for treatment of aggressive breast cancers.
Role of Chemokine Receptors in Breast Cancer Metastasis
Brett A. Premack, Ph.D.
University of California, Los Angeles; Jonsson Comprehensive Cancer Center
We need to understand more about how breast cancer cells become motile, particularly the ways that they enter and leave the lymphatic and blood vessels, which are the critical conduits to spread in the body. White blood cells are capable of moving out of the blood and lymph, and much is known of the specific cell surface proteins and soluble factors that regulate this process. These proteins and receptors are called chemokines. Although there is convincing evidence that chemokines and their cell surface receptors are involved in the growth and metastasis of some human tumors, the exact molecular nature of this involvement has yet to be elucidated. In human breast cancers, spreading to bone marrow and lung is characteristic of late stage disease in a large percentage of patients. In order to better understand this pathogenesis we will study the role of chemokines in breast cancer metastasis using molecular biology techniques and cell motility assays. Our basic premise is that because chemokines turn on the cellular machinery that produces directional migration of many cells, these same molecules may contribute to the metastasis of primary breast tumors.
To examine the possible presence and function of chemokines and their receptors in breast cancer, we will use new molecular assays (PCR-based), immunological (ELISA) techniques and physiological tools initially developed in our laboratory for the study of leukocytes. Thus, we will: (1) look for the mRNA expression of chemokines and receptors, (2) determine whether chemokine receptors are functionally coupled to calcium signaling pathways involved in breast cancer cell motility, (3) determine whether expressed chemokine receptors stimulate the migration of breast cancer cells, and (4) determine whether any of the breast cancer cell lines secrete chemokines which may function as autocrine (i.e., locally acting) motility factors. This study could uncover an autocrine motility loop, which might explain the basis for increased metastatic potential.
A real plus to our breast cancer-chemokine survey is that we will make use of a number of new anti-inflammatory and anti-viral chemokine therapeutics currently being developed in immunology research. If chemokine receptors are involved in cancer metastasis, it is quite likely that some of these newly developed drugs would also have anti-metastatic properties. One can envision a potent metastasis inhibitor based on controlling the motility of primary tumors. Such a drug could be a major new tool in the fight against breast cancer.
Innovative, Developmental and Exploratory Awards – Type II
A New Gene Regulation Factor, LMO-4, in Breast Cancer
Bogi A. Andersen, M.D.
University of California, San Diego
The etiology of sporadic breast cancers is multifactorial and is thought to involve stepwise mutations in several known oncogenes (tumor promoting) and tumor suppressor genes. The elucidation of novel pathways involved in regulation of proliferation and differentiation of breast epithelial cells has important implications for pathogenesis, prognosis and the identification of targets for therapeutic agents in breast cancer. We have recently cloned a novel gene regulation co-factor, LMO-4, which exhibits prominent expression in breast epithelial cells. Previously characterized members of this gene family have been shown to be required for normal blood cell differentiation and cause lymphocyte-type tumors. Consistent with a role in proliferation and/or early differentiation, the LMO-4 gene is highly expressed in proliferating epithelial cells of the breast. Data suggests that LMO-4 functions as a component of multiprotein complexes that regulate transcription. We hypothesize that LMO-4 participates in regulation of proliferation and differentiation in normal breast epithelial cells, and this may lead to altered patterns of gene expression that contribute to breast cancer.
To test this hypothesis, we plan to study in more detail the expression pattern of LMO-4 both during normal breast development and in breast cancers particularly with respect to cellular proliferation and differentiation. We plan to introduce both wild-type (i.e., the normal, non-mutated form) LMO-4 and an artificial LMO-4 repressor in the mammary glands of mice. Using mice that are genetically engineered (transgenic) we will be able to examine the effects of LMO-4 on mammary development, differentiation, and tumor processes. As a final goal, we will use the yeast two-hybrid technology to search for proteins that interact with LMO-4 and work with it in gene regulation. Specifically, we suspect that LMO-4 interacts with previously described transcription factors, either nuclear oncoproteins or tumor suppressors, known to be important for the pathogenesis of breast cancer.
These experiments should provide insights into how LMO-4 regulates cellular proliferation and differentiation in normal and neoplastic breast.
A New Model for Inflammatory Breast Carcinoma
Sanford H. Barsky, M.D.
University of California, Los Angeles; School of Medicine
Inflammatory breast cancer is an unusual type of the disease that is characterized by a very high degree of invasion into the blood and lymphatic vessels. We have established the first human model of this form of the disease, a transplantable inflammatory carcinoma xenograft model called MARY-X. This is a human tumor obtained from a patient with inflammatory breast carcinoma, which grows in mice similar to humans. This allows us to study the detailed molecular mechanisms that regulate inflammatory carcinoma. Our specific interest is the invasion of cancer cells into the blood and lymphatic vessels. MARY-X is extremely aggressive in this invasion process, and it even turns the skin of the mouse bright red just like inflammatory cancer does in humans.
We compared both MARY-X and MARY (the corresponding human cell line) with non-inflammatory breast carcinoma cell lines and xenografts for molecules including (1) angiogenic factors (molecules which cause blood vessel formation), (2) proteolytic enzymes (molecules which help cancer cells spread), and (3) adhesion molecules (molecules which help cancer cells stick to one another and to blood vessels) to determine differences that could account for the inflammatory phenotype. We found that angiogenic factors and proteases do not account for these differences. However, we noted a marked overexpression of MUC1 (a glycoprotein mucin found in the cell membrane of a variety of tumor types) and surprisingly E-cadherin (a cell-cell adhesion molecule). E-cadherin has been shown to be lost in most breast cancers rather than overexpressed, so our observation was quite surprising.
Based on these initial studies, we will pursue two aims designed to advance our knowledge of the mechanism of the inflammatory phenotype. First, we plan functional studies designed to examine whether MUC1/E-cadherin expression actually causes or contributes to the inflammatory phenotype. We will alter the expression of these proteins using both gene transfection and neutralizing antibodies and determine the effect on lymphovascular invasion in mice. Secondly, we plan a molecular comparison of inflammatory carcinoma vs. other non-inflammatory carcinomas using a technique of 'differential display' to identify those genes that are uniquely expressed or not expressed in inflammatory carcinoma.
Our goal is to gain further understanding the characteristics of inflammatory breast carcinoma and what sets this deadly disease of women into motion.
Molecular Structure of BAG-1: A New Protein in Breast Cancer
Kathryn R. Ely, Ph.D.
The Burnham Institute
Like normal breast tissues, many breast tumors depend on estrogens for growth and differentiation. Because of this, for the past three decades, endocrine therapies based on antiestrogens have been widely used to treat breast cancer. This treatment is often effective for patients, but with time the tumors become unresponsive to tamoxifen and the treatment fails. New approaches are needed that don't rely solely on antiestrogens. The focus of this project is BAG-1, a new protein that is linked to the estrogen receptor and makes breast cancer cells resistant to the growth inhibitory effects of tamoxifen. Breast cancer cells continue to grow and divide in the presence of tamoxifen, if BAG-1 is present. The goal of the present study is to generate a molecular image of BAG-1 using the methods of x-ray crystallography and nuclear magnetic resonance (NMR) imaging.
Our aims are to study a conserved domain of BAG-1 by NMR to gain information on functional properties that might prove useful in future drug design efforts. Previous work by our collaborators has served to isolate BAG-1, develop antibodies for purification and analysis, and understand the mechanism of its cellular functions. To this point, we have been able to produce BAG-1 as either the entire protein or the functional regions, and purify them in preparation for crystallization. A key long-term goal is to study BAG-1's interaction with another protein, celled Hsp70, which is a key link to hormone responsiveness. It is clear that BAG-1 is associated with hormone responsiveness, but the required structural features and how the smaller protein domains and accessory proteins, such as Hsp70, interact is unclear. The structure will reveal the precise 3-dimensional features of BAG-1 and will provide critical clues linking structure and function.
These pilot studies could serve as the first step in a drug discovery process for an alternate treatment of breast cancer. The molecular models may offer a new perspective to search for therapies for breast cancer. Since BAG-1 may contribute to treatment failure with tamoxifen or other antiestrogens, the BAG-1 molecule itself may represent a novel target for an entirely new treatment strategy.
Breast Cancer Cell Binding to the Endothelium<
Brunhilde Felding-Habermann, Ph.D.
The Scripps Research Institute
The existing paradigm is that breast cancer spreads primarily via lymphatic routes. However, in advanced stages of this disease, the colonization of preferred target organs such as bone, lungs, liver and brain evidently involves the blood stream. This contributes considerably to the morbidity and mortality in human breast cancer. Therefore, this study addresses mechanisms of hematogenous (via the blood) spread of breast cancer. We plan to investigate the adhesion receptors that support attachment of breast cancer cells to the endothelium of target organs under conditions that occur in the vasculature. Our approach is unique in that it addresses critical questions of tumor spread using conditions of actual blood flow in the body combined with metastatic tumor cells isolated from patients, and it is focused on specific candidate adhesion receptors on both endothelial cells and breast cancer cells.
The specific aims of this project are to (1) analyze the attachment of human breast cancer cells to endothelial cells of target organs (e.g., lung and bone) under blood flow conditions in vitro, (2) challenge our ideas in the intact vasculature in a living mouse, and (3) focus on the adhesion receptors integrin avb3 and ICAM-1 for their combined ability to support tumor cell arrest at the endothelium using specific monoclonal antibodies as neutralizing agents. To mimic the conditions in the vasculature as closely as possible, our experimental approach uses a novel in vitro flow system, based on fluorescence and confocal microscopy. We will then use intravital microscopy to examine these processes in an intact animal. For the isolation of circulating tumor cells from breast cancer patient blood, we will use special magnetic beads for cell concentration. Our experimental hypothesis is that fibrinogen, the major clotting protein in blood, may serve to mediate breast cancer cell attachment to the endothelium by virtue of its ability to cross-link avb3 present on tumor cells with ICAM-1 present on endothelial cells.
If the planned studies are successful, we will obtain definitive information about a role of avb3 and the ICAM-1 in the arrest of metastatic breast cancer cells at the endothelium of target organs. This information may impact potential new therapeutic strategies aimed at preventing breast cancer metastasis to organs that are involved in the majority of deaths from this disease.
Specificity of Ras Signaling in Breast Cancer
Janis H. Jackson, M.D.
The Scripps Research Institute
In breast cancer, the normal control mechanisms that regulate growth, differentiation and/or cell death become defective. These control mechanisms consist of regulatory proteins on the surface and an inside of cells that send signals to each other via complex protein pathways. Collectively, this is referred to as signal transduction. Breast cancer cells frequently contain regulatory proteins on the external surfaces, such as the Her-2 receptor and EGF receptor, that have become defective or are expressed at abnormally high levels. These defective/over-expressed receptor proteins send continuous signals to a regulatory protein called Ras, and these signals convert Ras from its inactive GDP-bound state to its active GTP-bound state. Breast cancer cells also frequently contain estrogen receptors, and when these receptors bind estrogen, they also send activating signal to Ras. Ras is located on the inner surface of cells, and once it has become activated, it sends activating signals to other regulatory proteins, and thereby initiating downstream signaling cascades that can promote breast cancer cell growth. Human cells contain four very similar Ras proteins, H-Ras, N-Ras, K-Ras 4A and K-Ras 4B, but it is currently unknown whether each or only one of these Ras proteins contributes to breast cancer cell growth. The purpose of our studies is to determine whether the Her-2, EGF, and/or estrogen receptors send activating signals to each of the Ras proteins or only one of the Ras proteins. For our experiments we will use breast cancer cells over-expressing the Her-2, EGF, or estrogen receptor. cDNAs encoding each of the four Ras proteins will be introduced into these cells by transfection. To determine whether the Her-2, EGF and/or estrogen receptors send activating signals to each or only one of the four Ras proteins, we will assess the relative amounts of GTP vs. GDP bound to each of the Ras proteins in the transfected cells. In addition, we will determine whether there are any differences in two downstream indicators of Ras activation (activation of MAP kinase and cell growth) in these cells.
Our studies are important, because demonstration that a single Ras protein contributes to breast cancer could provide the rationale for the development of inhibitors specific for that single Ras protein. These inhibitors could potentially help neutralize breast cancer growth in the approximately 30% of human breast cancer containing defective/over-expressed Her-2 or EGF receptors. In addition, these inhibitors might also be helpful in treating the approximately 50+% of breast cancers containing estrogen receptors.
Role of the EphB4 Receptor Tyrosine Kinase in Breast Cancer
Elena Pasquale, Ph.D.
The Burnham Institute
The formation of breast tumors is a multistep process in which a series of changes (gene mutations) occurring in sequence progressively transform normal cells of the breast into cells that multiply uncontrollably. Additional changes may occur that enable breast cancer cells to detach from the primary tumor and form metastases at distant sites in the body. Some of the genes that become mutated during breast cancer progression have been identified, but many likely remain unknown. It was recently discovered that a specific protein (receptor tyrosine kinase EphB4) is present at elevated levels in highly malignant human breast tumors. EphB4 is present in all the human breast cancer cell lines that have been examined. In transgenic mouse models of breast cancer, elevated levels of EphB4 were detected in the more aggressive undifferentiated (immature-looking) and metastasizing mammary tumors, but not in the less aggressive non-metastasizing and well differentiated (mature-looking) mammary tumors. Taken together, these data suggest that the EphB4 receptor plays a role in breast cancer, particularly in its more aggressive stages. EphB4 belongs to the Eph family of receptor tyrosine kinases. Developmental studies have shown that the Eph receptors, by regulating cell adhesion and cell movement in the embryo, are important for the proper organization and integrity of tissues. We have also recently identified a new protein, SHEP1, which may be turned on by Eph receptors, thus promoting breast cancer cell survival. Because tissue disorganization and abnormal cell adhesion, movement, and survival characterize the more advanced stages of cancer, the inappropriate functioning of an Eph receptor in breast tumor cells would be expected to make them more malignant.
We propose to study how changes that either promote or inhibit EphB4 signaling pathways affect the growth, invasiveness, and metastatic potential of human breast cancer cells. We will use nude mice as an animal model to study the properties of tumors derived from genetically engineered breast cancer cells. By taking advantage of a newly developed technology, the cells in which EphB4 signaling pathways have been altered will also be made fluorescent, and therefore will be easy to recognize. The proposed studies will be the first to explore the function of an Eph receptor in a human breast cancer model. The precise role of the EphB4 receptor in human breast cancer is still a matter of speculation. However, data demonstrating a correlation between EphB4 expression in breast tumors and the degree of malignancy, make a compelling case that the role of EphB4 in breast cancer should be investigated. The fact that the Eph receptors are different from most other receptor tyrosine kinases in that they regulate cell adhesion, migration, and probably cell survival rather than cell division, further supports the idea that EphB4, may have a distinctive role in the more advanced and deadly stages of breast cancer. If this hypothesis is correct, EphB4 could serve as a target gene for therapies to stop breast cancer progression and as a marker of tumors that require more aggressive treatments. Our studies will also provide information on whether SHEP1, a newly identified protein that likely propagates some of the EphB4 signals, could also be a marker for malignancy and a therapeutic target.
Targeting Breast Cancer Blood Vessels
Jan Schnitzer, M.D.
Sidney Kimmel Cancer Center
Breast cancer cells are 'hidden' behind the normal cells in the body, and it is difficult to directly target them in clinical settings. This thin barrier between the blood and the tumor is composed of endothelial cells, which serve to supply the tumor with nourishment and they later become a convenient avenue to spread in the body. It is appreciated that the tumor endothelial cells are different from normal vascular endothelial cells, but we know little about their specific proteins. Our interest is to gain more information about the tumor blood vessels, and use this information to generate monoclonal antibodies for immunotherapy. This approach would target the luminal surface (i.e., side of the cell facing the blood) of endothelial cells that feed tumor cells. Thus, our eventual strategy for immunotherapy of breast carcinomas is to target cytotoxic agents to the endothelial cells lining the tumor blood vessels, thus removing the tumor's blood supply Proteins at the luminal surface of the endothelium from normal tissue and from tumors of rat mammary carcinoma cells will be identified and compared by utilizing a novel membrane-isolation scheme that selectively isolates luminal endothelial cell plasma membranes from cancerous tissue. The proteins in these membrane fractions will be comparatively analyzed and characterized to create a very useful molecular map of the vascular endothelial cell surface. So far, in our preliminary studies, we have found several proteins selectively associated with the tumor endothelium. Specific monoclonal antibodies will be produced to these tumor endothelial proteins in order to target the vasculature of the tumor. Proteins of interest will also be purified for amino acid analysis to determine their identity. Our plan is to find numerous tumor-specific endothelial proteins that are not found in normal tissues. We will collect and assemble this information into a vascular endothelial cell proteonomic (VEP) map, which can be likened to either a library or database of potentially useful molecular targets for breast cancer immunotherapy.
Finally, those antibodies found to be specific for the tumor endothelium will be tested in animal models to see if they are effective in treating experimental breast cancer. Our experiments in this study are focused on the primary tumor, but we plan to extend our work to target the critical tumor vessels in distant sites of metastasis (e.g., lung). Thus, the potential of the VEP map and specific targeting antibodies will be the ability to use immunotherapy for advanced metastatic disease, which is usually resistant to current therapy options.
Spatial Control of Matrix Proteolysis in Breast Cancer
Alex Strongin, Ph.D.
La Jolla Institute for Experimental Medicine
The long-term objectives of our proposed research are to enhance understanding of the pathogenesis of breast cancer and to develop more effective interventions for treating this disease. There are strong correlations between breast cancer progression, invasion, metastasis and metalloproteinase activity in breast tumors. Metalloproteinases (MMPs) are the enzymes essential for the degradation of the matrix surrounding tumor cells that enable tumors to metastasize and grow in size. To control breast cancer, it appears necessary to regulate the MMPs expressed in most invasive breast tumors. In addition, tumors carry specific receptors capable of promoting the adhesion and invasion of breast cancer cells. The focus of the present study is to explore mechanisms by which MMPs can be localized to the cell surface of breast cancer cells to maximize their activity in facilitating cell migration. Our hypothesis is that the integrin receptor avb3 is the prime candidate for interacting with MMPs, called MT1-MMP and MMP-2.
Thus, our aims are designed to (1) understand how MMPs modify receptors of breast cancer cells, (2) identify the mechanisms and components capable of delivering MMPs to the discrete regions at the cell surface in an immediate proximity to these cell receptors, and (3) determine how we can employ this knowledge to design efficient inhibitors of tumor cell locomotion. These experiments will be performed by co-expressing MT-1-MMP and the integrin b3 subunit proteins in breast carcinoma cell lines. Using this approach we will be able to determine whether the integrin adhesion receptor and the MMP interact with each other and whether there are localized proteolysis effects leading to enhanced cellular movement.
These models will provide us with a challenging opportunity to discriminate and specify the effects of individual components involved in the locomotion of tumor cells. Understanding these mechanisms in should ultimately provide clues to facilitate the design of novel efficient inhibitors of focal proteolysis and breast cancer cell invasion.
How Does Endostatin Inhibit Angiogenesis?
Kristiina Vuori, M.D., Ph.D.
The Burnham Institute
It is now known that the growth and spread of solid tumor cancers, such as breast cancer, absolutely depends on the development of a tumor-associated vasculature by a process known as angiogenesis. Thus, there is considerable interest in finding ways to inhibit angiogenesis as an effective means for preventing tumor progression and metastasis. Our interest is an angiogenesis inhibitor protein called endostatin. It is a proteolytic cleavage product of type XVIII collagen and is one of the most potent angiogenesis inhibitors. Two important findings have increased our interest in endostatin. First, endostatin has been found to inhibit the subcutaneous growth of several tumor types in a mouse model, demonstrating that endostatin works on a wide spectrum of cancers. Secondly, repeated cycles of endostatin treatment have been shown to induce tumor dormancy, suggesting that endostatin treatment does not generate drug resistance. But, the molecular mechanism of endostatin function is not known. Our research is designed to investigate the relationship of endostatin function with certain key adhesion receptors present on endothelial cells.
Our preliminary results demonstrate that endostatin associates with two cell adhesion receptors, the avb3 and avb5 integrins. Significantly, these integrins have been previously identified as crucial molecules in controlling angiogenesis. Our hypothesis is that endostatin exerts its anti-angiogenic effects by interfering with the function of av-integrins in endothelial cells. This would disrupt endothelial cell attachment and a loss of tumor vessel function. In the first aim, we will identify the integrin binding site(s) in endostatin and characterize the significance of the integrin binding in endostatin function. Our preliminary results have identified three likely binding sites in the endostatin molecule. We plan to confirm and expand these observations by making mutant forms of endostatin in order to exactly identify the integrin-binding site. The second aim will be to characterize the effects of endostatin on endothelial physiological functions. Primary endothelial cells will be used as a model system, and the biological effects of wild-type and mutant forms of endostatin will be examined. Such cell processes as apoptosis (i.e., programmed cell death), proliferation, migration and endothelial tube formation will be studied.
Our research will expand knowledge of the structure-function relationship of endostatin, leading to possible active fragments for therapeutic development. This appears critical, since there have been reports of difficulty using the intact protein. It will also give insights into the cell biological and molecular pathways by which endostatin works on endothelial cells. Overall, these studies will take critical steps in the clinical use of endostatin in treating breast cancer.
GATA-3 Expression in Hormone Responsive Breast Cancer
Ronald Weigel, M.D., Ph.D.
Stanford University
The female hormone, estrogen, plays an important role in normal breast development and in the development of breast cancer in women. For reasons that are not entirely clear, some cancers retain their ability to respond to estrogen. Under these conditions the anti-estrogen, tamoxifen, has been shown to reduce the growth and spread of these cancers. The ability of cancers to response to tamoxifen is related to the presence of a protein in the cancer cells known as estrogen receptor (ER). ER is a protein that is present inside the cell of some breast cancers and which controls the ability of that cell to grow in the presence of estrogen or stop growing in the presence of anti-estrogens such as tamoxifen. When patients are treated with tamoxifen for long periods of time, cancer cells will develop that no longer respond to tamoxifen. Many of these cancers still make ER, which leads us to conclude that something in addition to ER is necessary for tamoxifen to work in a clinical setting. We have recently found another protein called GATA-3, which is made in almost all breast cancers that make ER. We know from other studies that GATA-type proteins are able to bind to ER, thereby altering the function of these two proteins. Drugs such as tamoxifen can break apart GATA and ER thus allowing the GATA protein to turn on other genes in side the cell. Our novel finding, therefore, suggests a new mechanism of tamoxifen action and offers the possibility of understanding why some patient's tumors no longer respond to tamoxifen.
The experiments planned will allow us to understand what GATA-3 is doing in breast cancer cells and how tamoxifen alters the function of GATA-3. We know from other studies that GATA-3 turns other genes on inside cells. We will first define which genes are regulated by GATA-3 in a hormone responsive breast cancer cell line. Genes controlled by GATA-3 will be found by comparing genes that are turned on when GATA-3 is put into cells and by finding genes that are turned off when GATA-3 is eliminated. We will then determine if the genes controlled by GATA-3 can be turned on and off with the drug tamoxifen. We expect to show that the ability to switch genes on and off with tamoxifen or estrogen requires both ER and GATA-3 proteins. Ultimately, these experiments may provide a way to help us predict which women will be helped by tamoxifen treatment and may offer new approaches to treat women whose tumors become resistant to tamoxifen therapy.
The Regulation of p53 Activity in Breast Cancer
Yang Xu, Ph.D.
University of California, San Diego
Hereditary breast cancers account for about 5-10% of all breast cancers and a large percentage of them are early-onset breast cancers. Mutations of a number of tumor suppressor genes, including BRCA1, BRCA2, p53 and PTEN/MMAC1, are associated with the hereditary breast cancers. Recent studies have suggested that p53 mutations are necessary for tumor formation in BRCA-deficient tumors. In addition, p53, the most commonly mutated tumor suppressor in various human cancers, is also mutated at a high frequency in non-hereditary (sporadic) breast cancers. Therefore, understanding the regulation of p53 function in tumor suppression is essential to design new therapeutic strategies to treat this disease.
It appears that p53 can have two roles in tumor suppression, leading to either cell-cycle arrest or apoptosis (programmed cell death). Both p53-dependent functions appear to be involved in maintaining the integrity of the genome, such that excess DNA mutations leading to cancer will not accumulate. Normally p53 is present in an inactive form and at very low levels. But, with DNA damages (e.g., radiation or chemotherapy), p53 activity is induced. Our studies are aimed to employ genetic approaches to address the involvement of phosphorylation, which is the addition of phosphate groups to specific Ser/Thr sites in the protein structure, in p53 activation for both gene regulation and apoptosis functions.
We will employ new genetic approaches to introduce mutations of various p53 phosphorylation sites and use embryonic stem cells to study the effects of these changes. An important advantage of this assay is that the mutant p53 is expressed under the control of its own gene promoter. In addition, the mutant embryonic stem cells can differentiate into other primary cell types that we can use to address the question of apoptosis. In conclusion, these mutant embryonic stem cells will provide an ideal physiological model system to study the effects of these p53 mutations on the p53 responses to various DNA damages. Our eventual goal is to screen breast cancer cells for not only the p53 mutations but also the defects in these signaling pathways involved in the p53 phosphorylation. But first, we need the results from this study to determine which phosphylation sites appear linked to events critical to cell cycle arrest and apoptosis. When these issues become resolved we will be in a better position to deal with problem of failed therapies for breast cancer.
New Investigator Awards
A Role for RAD51B in Breast Cancer
Joanna S. Albala, Ph.D.
Lawrence Livermore National Laboratory
The development of breast cancer is associated with defects in critical cellular pathways, such as those involved with DNA repair. The hereditary breast cancer genes, BRCA1 and BRCA2, are known to be involved in one such pathway. Another critical member of this DNA repair pathway, RAD51, has been shown to interact with the BRCA1 and BRCA2 proteins. These proteins have also been co-localized in the nuclei of malignant breast cell lines. Taken together, these studies indicate a role for RAD51 in breast cancer. We have discovered a novel gene, RAD51B, whose DNA sequence suggests a function analogous to RAD51 and is therefore believed to be involved in these processes. DNA repair defects in these genes may lead to the accumulation of genetic mutations and chromosome recombination events that characterize advanced breast cancer.
The screening of a public database for RAD51-like genes led to the identification of a partial unknown sequence from a breast cDNA library, which was later characterized as RAD51B. Isolation and molecular analysis of the RAD51B gene showed that this gene is widely expressed and most abundant in tissues active in recombination. In normal tissues, the RAD51 family functions to preserve chromosomal integrity and promote genetic diversity through chromosomal recombination during meiosis in the egg and sperm. It is likely that in cancer cells, disruption of the RAD51B gene within this pathway will lead to enhanced chromosomal instability and therefore accelerate genetic changes leading to defects in cell function and morphology.
The specific aims of this research project are to: (1) express and and purify recombinant RAD51B and define its localization pattern in normal and malignant breast cancer cell lines; (2) characterize the interactions between RAD51B and the breast cancer gene products, BRCA1 and BRCA2; and (3) isolate and characterize novel binding partners of the RAD51B protein. The approach to identify the native RAD51B will be to isolate protein from cellular extracts derived from both normal and malignant breast cell lines. Newly produced and optimized RAD51B antibodies will be used in these studies. Specific interactions between RAD51B and the BRCA1 and BRCA2 proteins will be examined using yeast two-hybrid analysis. We aim to identify and isolate novel binding partners of the RAD51B protein using immunoaffinity chromotography techniques.
The thrust of these studies is to identify protein factors that interact with RAD51B and thus provide insights into the biological role of the RAD51B protein in mediating the genetic defects that underlie breast carcinogenesis.
Characterization of Hag-2 and Its Role in Breast Cancer
Devon A. Thompson, Ph.D.
Stanford University
All breast tumors, from either biopsies or excisions, are examined for the presence of a protein called estrogen receptor. This helps clinicians to evaluate the prognosis and most suitable course of treatment for the patient. Many tumors that contain estrogen receptor (ER+) prove to be responsive to hormone therapy, whereas those tumors that do not contain estrogen receptor (ER-) respond poorly to hormone therapy. Overall, ER+ breast cancers are a better target for hormone therapy, often using the anti-estrogen drug tamoxifen.
We have discovered a new gene, called hAG-2, that appears to be operative only in those breast tumors that are ER+. We are examining many aspects of hAG-2 in efforts to understand how and why it is present, together with ER, in breast cancer tumors. This is important, because even though ER+ breast cancers respond to hormone therapy, often these therapies fail or resistance develops. Thus, more information, targets for therapy, and potential clinical biomarkers are needed for women with ER+ breast cancers. The overall objective of this research project is to delineate the biological role of hAG-2 and to ascertain the significance of hAG-2 as a marker for mammary and breast tumor differentiation. Our specific aims include, (1) examining clinical breast tumor samples for hAG-2 amounts, particularly correlated with tumor grade, ER status, and stage, (2) studying hAG-2 expression during mammary development in the mouse, and (3) producing hAG-2 as a recombinant protein and generating antibodies for detailed cell localization and other studies. More information on factors that control hAG-2 in breast cancer will clarify the significance of ER as the most important clinical marker of the disease and focus (and failure) of current therapy. The association of hAG-2 with ER implicates it as a key player in breast tumor biology.
We anicipate that hAG-2 will emerge as an important factor for breast cancer tumor development, and perhaps mammary development. Ultimately, knowledge at the molecular level will be pivotal to designing intelligent cancer treatment strategies.
Postdoctoral Fellowship Awards
Role of a DNA Damage Response Gene in Breast Cancer
Eric Brown, Ph.D.
California Institute of Technology
Numerous studies have demonstrated an increased incidence of breast cancer in women exposed to therapeutic and occupational levels of radiation. This radiation mutates cellular DNA, which ultimately leads to breast cancer in these women. However, besides these unusual circumstances, DNA damage occurs continually in all individuals by means of normal metabolic processes, like the breakdown of food into energy. Since damaged DNA is widely considered to be the dominant force in the generation of cancer, the efficiency with which cells protect the integrity of DNA is imperative for the prevention of cancer in all individuals. Consistent with this analysis, the elimination of genes that repair damaged DNA or prevent mutated cells from growing is well correlated with the generation of breast cancer. For example, prognosis is poor for breast cancer patients with tumors that harbor mutations in p53, a protein that normally prevents mutated cells from dividing. In addition, people with mutations in the breast cancer associated genes, BRCA1 and BRCA2, have a 200 fold greater risk of breast cancer.
We have evidence for the likely involvement of a gene called ATR in responses to damaged DNA. ATR is related to the ATM tumor suppressor gene and is the human equivalent of genes used in other organisms for maintenance of DNA integrity. The similarity of these genes and recent studies indicate that ATR is potentially involved in regulating genes known to be required to prevent mutated cells from multiplying, like p53. In addition, I provide preliminary evidence that further substantiates a link between the function of BRCA1 and ATR. To explore the role of ATR in responses to damaged DNA, I plan to eliminate ATR from fully developed cells. Thus, if ATR is necessary for regulating BRCA1 for example, then when it is eliminated BRCA1 should be unresponsive to damaged DNA. In addition, the method I plan to use to eliminate ATR is novel and would prove useful for studying the function of genes like BRCA1 and BRCA2. Finally, I have an experimental strategy to identify yet undiscovered genes that normally function in preventing breast cancer.
Together, these studies will lead to better prevention and treatment of breast cancer. For example, 80% of all breast cancer is of late onset and late onset breast cancer is often aggregated in families. It is therefore reasonable to hypothesize that genetic predisposition causes the development of breast cancer in these cases. Potential candidates for these genes include molecules that regulate DNA-damage responses like ATR. In addition, knowledge of these mechanisms may not only lead to a better understanding of the predisposition to and prognosis of breast cancer, but also to better treatments. It is conceivable that treatments for breast cancer might one day be formulated and chosen based on the genetic characteristics of both the tumor and the patient. It seems likely that ATR and the molecules it regulates will be among these genes.
COX-2 and Apoptosis Regulation in Breast Cancer
Youngsoo Kim, Ph.D.
The Burnham Institute
The growth of tumors is caused by imbalances between the rates at which cells are produced through cell division and the rate at which they die through a natural cell death process known as programmed cell death (also referred to as apoptosis). Defects in the apoptosis pathway allow tumor cells to survive for prolonged periods of time, accumulate genetic errors, and live in a suspended state that permits metastatic spread. Problems in programmed cell death mechanisms also contribute to therapy resistance, making tumors more difficult to kill with radiation or chemotherapy. It is important therefore to understand why tumors become refractory to programmed cell death and to devise strategies for restoring proper function to cell death pathways in tumor cells.
Recently it has been discovered that aspirin-like drugs can restore proper function to cell death pathways in some types of tumors, including breast cancers. Aspirin-like drugs function primarily by inhibiting an enzyme in cells called cyclooxygenase-2 (COX-2). The levels of COX-2 are elevated in aggressive breast cancers. Higher levels of COX-2 have been reported to make some kinds of cancer cells resistant to programmed cell death by turning on genes that promote cell survival. COX-2 inhibitory drugs can shut off these cell survival genes and make it easier to kill tumor cells. One of the roles of COX-2 in tumor development is its positive effect on the survival of tumor cells. Thus, the efficacy of aspirin-like drugs in chemoprevention and treatment of tumors is also in part due to their abilities to induce apoptosis.
The goal of this project is to provide insights into the role of COX-2 in breast cancer. We will explore whether COX-2 inhibitors can shut off survival genes in breast cancer cells, and we will study how COX-2 affects the programmed cell death machinery of breast cancer cells. Some specific aims include (1) studying the gene regulation of COX-2, (2) creating COX-2 overexpressing cells and developing methods for blocking COX-2 expression, and (3) studying the influence of PPARs (a nuclear steroid hormone receptor family) and COX-2 in apoptosis. In addition, we will explore why COX-2 levels are elevated in some breast cancers, so that the relation of COX-2 to breast cancer progression can be understood.
Understanding the mechanisms by which COX-2 contributes to cancer development will reveal to what extent this enzyme is an important target in the chemoprevention and treatment of breast cancer.
Proteolysis of Cyclin E in Normal and Malignant Breast Cells
Heimo Strohmaier, Ph.D.
The Scripps Research Institute
In order for a breast cell to divide, it must pass through an ordered progression of control, check-points, called the cell cycle. This cell division cycle is composed of growth phases (G1 and G2), chromosome duplication phase (S), and a cell division phase (M), which are temporally G1, S, G2, and M. Unfortunately, in breast cancer cells, the control mechanisms can become defective, which cause cells to constantly proliferate.
The key checkpoints are regulated by proteins called cyclins, and an associated group of regulatory proteins called cyclin-dependent kinases (CDKs). In normal cells, periodic synthesis and destruction of cyclins is essential for ordered cell cycle passage. In normal cells, the decision is often to leave G1 and the move to a quiescent state, called G0. But in breast cancer, the decision of the cell, which might be based on defects in cyclins and CDKs, is to go directly into S (DNA replication) and proceed through cell division. A main feature of cyclins is their temporal nature in the cell. Our interest is in two elements of this process, cyclin E and its partner CDK2. Previous work has indicated that defects in synthesis or regulation (degradation) in these proteins are key to unwanted breast cancer cell division. Yeast is an excellent model to study the cyclins and CDKs, since their function has been conserved in evolution.
Our goal is to identify the cellular factors that are involved in cyclin E degradation. Recently, the components that target yeast G1 cyclins for ubiquitin-dependent degradation were identified. Ubiquitin is a common method used by cells to tag proteins, so that they can be removed by proteolysis. Human cyclin E is functional as a G1 cyclin in yeast where it is also ubiquitinated and degraded. Thus, we will employ the yeast model system to identify the elements of the pathway that are responsible for cyclin E degradation. In breast cancer cells we will study a panel of 40 samples to determine the amount and activity of cyclin E. Next, using SSCP analysis, we will test whether cyclin E might be mutated in breast cancer to alter its rate of degradation. Finally, we will examine the degradation pathway of ubiquination to determine if there are defects in breast cancer.
This knowledge will help us to understand the role of cyclin E and other cell cycle regulators. Ultimately, this will potentially offer new approaches for diagnosis, therapy, and prevention of breast cancer.
A Novel Inhibitor of Breast Tumor Angiogenesis
YingQing Sun, Ph.D.
The Burnham Institute
Tumors cannot grow without a supply of nutrients and oxygen. These are only provided to the interior of a tumor if new blood vessels grow into the tumor mass, a process called angiogenesis. In order for angiogenesis to occur, new endothelial cells (the cells that line blood vessels) must tunnel into the tumor mass and then assemble into a blood vessel. Endothelial cells use a type of enzyme, called a matrix metalloproteinase (MMP), to digest proteins surrounding the tumor mass. This allows for endothelial cell migration. In this project, we will examine a novel mechanism associated with angiogensis and develop this information towards new antagonists of this critical process.
Our hypothesis is that a protease involved in tumor angiogenesis, MMP-2, binds to an adhesion receptor on the endothelial cell, called the avb3 integrin. This binding would facilitate angiogensis by localizing and concentrating the enzymatic function of MMP-2 to more effectively allow cell migration. We have designed a series of experiments to validate our hypothesis, develop additional information, and work towards developing novel peptide-based inhibitors useful in blocking angiogenesis. An important consideration is that the binding site on avb3 differs from the site that the integrin receptor uses for normal cell adhesion. Some approaches to be used in these studies include, (1) binding studies between the C-terminal PEX region of MMP-2 with avb3 and other related integrins, (2) comparative studies of PEX-avb3 binding in comparison to other avb3 adhesion functions, (3) constructing molecular chimeras of b3 with other integrin b-subunits to define the site of MMP-2 interaction, and (4) expanding our findings to test novel inhibitory peptides, which is the first step in therapeutic development.
Our ultimate goal is to test our inhibitors first on proteins and cells, and later as inhibitors of angiogenesis in breast tumor models using experimental animals. Since our project involves a novel approach and hypothesis to consider this issue, we anticipate that the results will lead to new strategies and compounds for halting tumor angiogenesis and treating breast cancer.
