Prevention
For obvious reasons, prevention of breast cancer was selected as a high priority research area. The Breast Cancer Research Council considered the possible scope of prevention research and chose to encourage submission of research proposals aimed at identifying and eliminating causative agents, or therapies designed to prevent clinical manifestations of breast cancer. Since human clinical cancer prevention trials require large amounts of money and large numbers of subjects to achieve meaningful results, the Breast Cancer Research Council believes these types of trials are beyond the scope of the Program. Rather, the Council chose to focus on funding innovative, targeted research proposals that, if promising, could be continued with funding from other agencies and serve as examples nationwide.
There has been nationwide interest in the chemoprevention of all forms of malignancies including breast cancer. Federal programs that support the vast majority of breast cancer research (e.g. National Cancer Institute, Department of Defense) have focused on chemoprevention using hormonal strategies, particularly antiestrogens (e.g., tamoxifen), as well as high doses of selected vitamins (e.g., retinoids). To avoid duplicating these funded efforts, the Council encouraged innovative and non-conventional proposals spanning three broad areas of prevention research:
- development of new biologics (e.g., cytokines, antibodies, vaccines, enzymes) and natural compounds (e.g., phyto-estrogens) that might serve as preventative therapies;
- characterization of novel cellular mechanisms (e.g., integrins, growth factors and their receptors, telomerase) to be targeted by potential preventative agents; and
- identification of social and psychological strategies (e.g., behavior modification, emotional influences) that might also serve to prevent breast cancer.
The Council was pleased with the overall quality and variety of submitted breast cancer proposals covering the full range of prevention research, including the three areas specifically encouraged. The following nineteen proposals were awarded as the most important and innovative; and these awards are now funding a significant group of breast cancer prevention investigators throughout the state's public and private university campuses, many of our smaller nonprofit research organizations and foundations, and within California biotechnology companies.
ABSTRACTS
Growth Inhibition of Breast Cancer Cells by Interferons
Ke Shuai, Ph.D.
University of California, Los Angeles
Interferons (IFNs) have antiproliferative activities and can inhibit the growth of many tumor cells including breast cancer cells. Unfortunately, clinical trials using IFN as a single drug for breast cancer have not been satisfactory due to a wide range of side effects resulting from the usage of high doses of IFNs. However, it has been shown that the strategy to use tamoxifen in combination with IFN can greatly enhance the effects of tamoxifen on breast cancer. Based on our most current understanding of the molecular basis of IFN action, we have recently discovered a protein molecule which can greatly enhance the antiproliferative effects of IFNs. The overall goal of this project is to study the effects of this protein molecule on the growth inhibition of breast cancer cells. We will also test our hypothesis that this protein molecule can greatly enhance the antigrowth effects of tamoxifen on breast cancer cells. This research will lead to the design of rational preventative strategies for breast cancer employing IFNs.
To study the effects of this molecule on the growth inhibition of breast cancer cells, we will first introduce this protein into breast cancer cells using a system in which we can control the level of this protein. We will then analyze the growth of breast cancer cells in the presence of different levels of this protein molecule. The optimal concentrations of IFNs for growth inhibition of breast cancer cells in the presence of this protein will also be determined. Next, we will investigate the possibility that this protein molecule can greatly enhance the antipro-liferative effects of tamoxifen on breast cancer cells. The growth inhibition of breast cancer cells by tamoxifen in the presence or absence of this protein molecule will be determined and analyzed. We will also determine the potential effects of this protein molecular on the level of estrogen receptor by Northern blot and Western blot analysis.
Prevention of Breast Cancer by Blocking Integrin Function
Jeffrey W. Smith, Ph.D.
La Jolla Cancer Research Foundation
The primary danger in breast cancer is the spread of the tumor to other organs in the body. Tumor cells use adhesion molecules to migrate, or in essence to “crawl”, to other regions of the body. If the activity of such adhesion molecules could be inhibited, then the metastasis of breast cancer could be considerably slowed and perhaps eliminated. Thus, the identification and targeting of adhesion molecules involved in breast cancer should be a high priority.
The central hypothesis of this grant is that adhesion molecules called integrins are key mediators of the metastasis of breast cancer. Integrins were so named because they provide an “integral” link between the inside and outside of the cell. Integrins are produced in all normal cells, but when produced at improper times or locations, like in breast cancer, integrins change cell behavior. We hypothesize that (1) in breast cancer integrins are required for the physical migration of tumor cells to distant organs, (2) that integrins are involved in the changes in gene function within the tumor cells as the tumor cell encounters new environments during metastasis and (3) that inhibitors of integrins can be applied to stop the metastasis of breast cancer.
As the first step in the project, the integrin adhesion molecules that are involved in the migration of breast cancer cells will be identified. Data in the literature correlate the production of integrins possessing the av subunit with invasive breast cancer. This information is an excellent start, but provides only a correlation. We aim to determine if there is a causal relationship between the presence of av-integrins and invasive breast cancer. In the initial phase of the project the ability of each of the four known alpha-v- integrins to enable tumor cell migration and tumor metastasis will be measured. Results from these experiments will be the first measure of the ability of these adhesive molecules to increase tumor cell motility and metastasis. The integrins that are found to cause tumor invasion will be designated as targets for inhibition of metastasis.
The progression of breast cancer is known to be the result of a series of genetic changes. In the second phase of the project we will examine the possibility that integrin adhesion molecules actually signal the cell to begin these genetic changes. Such a hypothesis is suggested because the integrins mediate the physical contact between the cell and its environment. As the tumor cell migrates to different locations, the integrin contacts different matrices. We hypothesis that integrins “sense” the environment of the tumor cell and signal corresponding genetic changes that could actually feed the fire, initiating further aberrant genetic alterations in the tumor cell.
The final phase of the study will be a test to determine whether two powerful inhibitors of alpha'v integrins can block the metastasis of mammary carcinoma in vivo. One inhibitor is a small organic molecule that binds the adhesive site on the alpha-v integrins. The other inhibitor is a human antibody that we have engineered to inhibit the adhesive activity of alpha-v integrins. Results from these experiments will indicate the potential for blocking breast cancer metastasis by interfering with integrin function.
EGFR Structure for Drug Design Targeted to Breast Cancer
Chung-leung Chan, Ph.D.
University of California, San Diego
Protein tyrosine kinases are a family of biomolecules that play crucial roles in maintaining a normal balance in cell growth and development. Two members of this family, EGFR and erbB-2, are particularly important in breast cancer because increased production frequently occurs in breast cancer. Tumor aggressiveness and patient outcomes can be directly correlated to their production levels. Drugs, which can control their activity, are able to stop the development of breast tumors and restore normal breast cell growth. However, the selectivity of this family of drugs is quite limited and often leads to serious side effects. Research proposed in this post-doctoral fellowship application addresses the fundamental issue about how to design specific drugs to prevent the progression of breast cancer. This proposal outlines several strategies that will allow us to examine the structure of EGFR in detail. The structural features that determine drug specificity will be identified. As the factors which control this specificity in EGFR begin to unfold, we will be able to design drugs with selective inhibitory effects on EGFR. Such progress may lead to specific drugs for controlling the growth of breast cancer cells.
Regaining Control over Breast Cancer Cells
Michael Karin, Ph.D.
University of California, San Diego
Early in life most cells divide to increase the mass of the organism; at that point they are “undifferentiated,” because they do not express specific traits. In response to specific signals, cells stop dividing and start expressing specific traits, thus they become differentiated. In an adult breast, most of the cells are differentiated as either fat cells or supporting tissue. A few undifferentiated epithelial cells divide during each menstrual cycle; some of the new cells differentiate as milk producing cell precursors. Without pregnancy hormones, the differentiated cells die. By contrast, breast cancer cells continue to divide and none “differentiate” to milk producing cells. Normal cells divide in response to “growth factors” which are “messenger” molecules from other parts of the body. Breast cancer cells also produce their own growth factors.
Undifferentiated cells recognize and respond to the growth factors through proteins called receptors embedded in their cell membrane. Cancer cells also produce excessive amounts of receptor proteins. One such protein that is over-produced in 25% of human breast cancer is called ErbB2.
Recently a small protein that binds ErbB2, called heregulin, was found; intriguingly, the effect of heregulin is inconsistent and difficult to predict. When applied to certain breast cancer cell lines, proliferation stops and differentiation is induced. However, when applied to other cells proliferation is stimulated. Thus, heregulin acts as a growth factor. We propose to study the molecular basis for this variability. This may allow the design of strategies for preventing disease progression by allowing heregulin to induce differentiation in a highly consistent and efficient manner.
The chemical signals generated by ErbB2 once it is bound to heregulin are transmitted to the cell's genetic machinery located in its nucleus through specialized enzymes called Mitogen Activated Protein Kinases (MAPKs). These enzymes transmit different signals that tell the cell to either divide, differentiate or commit suicide and die. This plethora of responses is made possible because there are three different types of MAPKs called ERKS, JNKs and p38/Mpk2. The exact biological functions of these enzymes are not well established. Therefore, we propose to examine the effects of heregulin on the three different enzymes in different types of breast cancer cells where it can induce either proliferation or differentiation. We propose that the exact cellular response to heregulin is determined by a “code” of MPK activation. We will try to “break” and interfere with this “code” through the selective inhibition of the enzymes described above. This, we hope, will allow different types of breast cancer cells to mount a consistent and predictable differentiation response to heregulin, thus stopping tumor growth.
Estrogen Receptor Regulation in Breast Cancer
Lisa A. McPherson, Ph.D.
Stanford University
In breast cancer, the majority of tumors found in postmenopausal women contain estrogen receptor while tumors in younger women often lack this protein. Estrogen receptor is a protein normally found in various reproduction related tissues such as the breast and uterus. When estrogen receptor binds estrogen, it becomes activated and can interact with the genome of the cell, resulting in the activation of selected sets of responsive genes. This results in changes in the synthesis of specific RNA's and proteins involved in the regulation of cell proliferation, differentiation and physiologic function. Although normal breast tissue also makes estrogen receptor, the amount of this protein produced in positive breast carcinomas is significantly higher. This may account for some of the differences seen in the abnormal growth of various tumors and tumor cell lines when compared to normal breast tissue development. Breast carcinomas that do not make estrogen receptor tend to recur earlier than estrogen receptor-positive tumors and are also more aggressive, independent of any effect of hormonal therapy. Elucidating the mechanism by which the production of estrogen receptor is controlled in these tumors will lead to strategies by which the growth of aggressive estrogen receptor-negative tumors could be controlled.
Recent experiments have indicated the existence of a protein, Estrogen Receptor Factor-1 (ERF-1), which is involved in the regulation of estrogen receptor production and which is only made in estrogen receptor-positive breast and endometrial carcinomas. In addition to regulating estrogen receptor, it is possible that ERF-1 also regulates other cellular genes critical to breast cancer. We hypothesize that ERF-1 is involved in maintaining the characteristics associated with estrogen receptor production in breast carcinoma. This study seeks to determine the protein sequence of ERF-1 and to study the mechanism by which it regulates estrogen receptor production in breast cancer cell lines. The identification of ERF-1 could aid in the development of gene therapy protocols to stop the progression of tumors lacking both ERF-1 and estrogen receptor.
Vitamin D and Breast Cancer Prevention: Cell Death vs Growth
Valerie M. Weaver, Ph.D.
Lawrence Berkeley National Laboratory
Breast cancer occurs as a result of a transition of normal breast epithelial cell behavior to that of uncontrolled cell growth. A major factor in this transition is the gradual loss of the normal cellular balance between cell division and cell death. An important function of the genes in a normal breast cell is to control the appropriate time for the cell to die. Such cell death balances the growth which occurs due to normal cell division. In all non-menopausal women there is a sequential burst of cell division followed by a tightly controlled round of cell death with each menstrual cycle. We have found that signals from the extracellular matrix (ECM, an interwoven network of proteins which is in contact with these breast epithelial cells) give not only strength and support but also information to these cells. Thus the ability of breast cells to respond correctly to these messages is critical for maintaining the normal cell balance. In breast cancer the genes of the breast epithelial cell are altered such that these cells are now unable to respond appropriately to breast tissue ECM-derived signals. There is reason to believe that the active compound of vitamin D may act on breast cells to regulate the genes required for this responsiveness. We hypothesize that vitamin D may reestablish the normal cell growth and death cycle and that these effects may be mediated through critical cell-ECM signals. Regardless of the mode of action, however, vitamin D may be effective in assisting with both the prevention of primary breast cancer and its recurrence following therapy.
We have access to a unique human breast cell line which mimics breast cancer progression as it happens in the body. In addition we have the expertise with a novel ECM assay which allows the reconstruction of the normal breast cell environment in culture. Using this system I will examine the ability of breast epithelial cells to respond appropriately to signals from the ECM as they undergo transition from normal to cancerous behavior. Emphasis will be placed on their ability to undergo appropriate cell death. After establishing when the critical changes occur in the progression series I will study the ability of vitamin D to rescue this aberrant behavior. Finally I will begin studies to determine what crucial factors are important for this altered responsiveness by isolating vitamin D responsive genes. The validity of these results will be determined by using clinical samples of normal and cancerous human breast tissue. These results will have important consequences for understanding the development of both primary and recurrent breast tumors. They should also provide information for the development of better diagnostic techniques.
The Breast Tumor Suppressor Function of Hyaluronidase
Robert Stern, M.D.
University of California, San Francisco
The enzyme Hyaluronidase appears to interfere with metastasic spread of breast cancer through three mechanisms. Two of the requirements for cancer cells to metastasize are that they must become motile, and that they must have a space into which to move. Hyaluronan (HA) accomplishes both of these tasks. Hyaluronan is a carbohydrate polymer on the surface of cancer cells, that hydrates, expands, and opens up spaces around cancer cells enabling them to invade surrounding tissues. It is also involved in the development of motility. Hyaluronidase, the enzyme that breaks down HA, eliminates this mechanism for cancer cell spread. Hyaluronidase appears to have other direct effects on cancer cells. The receptor for HA, CD44, functions as an anchor to bind HA to the cancer cell surface. CD44 comes in a variety of forms, a short form found on normal cells, and longer variants that occur on motile cancer cells. A portion of CD44 protrudes through the surface membrane into the cell cytoplasm, where it interacts with the cytoskleleton, the machinery for cell movement. Experimentally, inserting this longer cancer form of CD44 into benign tumor cells changes them into cancer cells that aggressively metastasize. Hyaluronidase eliminates the cancer cells' ability to move, by converting the long cancer form of CD44 to the short “normal” immobile form. Remarkably, hyaluronidase also causes established breast cancers to shrink.
In mice with human breast cancer, inoculation of hyaluronidase i.v. shrinks the tumor to half its size in four days. If malignant cells cannot maintain motility and thus invasiveness, and if the tumor cannot progress or grow, it apparently must undergo regression. This third function of hyaluronidase is not understood, but may follow from the first two functions. Hyaluronidase may also prevent progression of human breast cancer, and it does not have the devastating toxic side effects of other anticancer agents. We will vary injection schedules, and modify the enzyme chemically, to stabilize activity, in order to maximize the anticancer effect in mice, in preparation for clinical trials. Simultaneously, we will examine breast cancer cells in culture using molecular and cell biology to determine precisely how hyaluronidase regulates this alternative splicing of CD44. We postulate that HA regulates its own receptor. When HA bound to CD44 is present outside the cell, tags are placed on the intracellular portion of CD44. We have identified these specific tagging enzymes or phosphokinases, and when they are inactivated by techniques involving antisense or mirror image DNA, CD44 converts to its short form, as CD44 does in response to hyaluronidase. Understanding these events in the regulation of CD44 will make it possible to develop a new generation of anticancer drugs that prevent the metastases associated with breast cancer progression.
Breast Cancer Chemoprevention by Vitamin D Plus Retinoids
Marcia I. Dawson, Ph.D.
Stanford Research Institute International
Vitamin D3 and the retinoic acid family (oxidized forms of vitamin A) inhibit the proliferation and growth of human breast cancer cells, including the vascularization process in tumors, which is necessary for the tumor to maintain its growth. Therefore, these compounds have outstanding potential as chemopreventative agents to inhibit the recurrence and spread of breast cancer, provided that their toxic side effects can be reduced. Preliminary breast cancer cell growth inhibition experiments indicate that combinations of these substances will permit lower effective doses than would be required if each agent were used alone. Therefore, side effects should be reduced. We will investigate the interactive molecular mechanisms of action of vitamin D3 and several different retinoids in inhibiting breast cancer cell growth and proliferation.
These retinoids show a range of activity profiles that include such diverse effects on cancer cells as selectively inducing the synthesis of proteins involved in normal growth processes, inhibiting the synthesis of other proteins responsible for cell proliferation, and initiating the sequence of events that lead to tumor cell death. We propose to evaluate the effects of these retinoids alone and in combination with vitamin D3 on breast cancer cell growth under conditions in which cells become attached to surfaces (indicative of less likelihood of invading other tissues) and under conditions in which cells grow without the need for attachment (indicative of more invasive cancers). Our preliminary results indicate that both agents that mimic retinoid action and agents that block retinoid action inhibit the growth of cells under conditions requiring attachment, but that only agents that mimic retinoid action inhibit the growth of cells under conditions not requiring attachment. The growth of cells under these latter conditions correlates with their ability to grow as tumors and metastasize. We will also evaluate the effects of these combinations on breast cancer cell migration and invasion.
To assess the effects at the molecular level, we will evaluate these retinoids alone and with vitamin D3 on the levels of two markers of cell proliferation, epidermal growth factor and the gene c-myc, which is over-active in cancer cells. Our ultimate goal is to devise new strategies for the chemoprevention of breast cancer, including the prevention of either its recurrence or its further progression.
Prevention of Breast Cancer Tumor Growth by Retinoic Acid
Yi Liu, Ph.D.
La Jolla Cancer Research Foundation
A substantial body of data has demonstrated that retinoids, the natural and synthetic vitamin A derivatives, are effective in inhibiting the proliferation of breast cancer cells. Currently, retinoids are being used in clinical trials against breast cancer. However, the anti-proliferative effect of retinoids is most frequently observed in hormone-dependent human breast cancer cells, and this effect appears to diminish in hormone-independent cells during the progression of breast cancer. The molecular mechanism of growth inhibition by retinoids is currently unclear. In the proposed project, the molecular mechanism of the retinoid growth inhibitory effect will be studied. Results from this study may provide a molecular basis for developing more effective and potent retinoids in the prevention of breast cancer, and may also lead to early detection and valuable diagnostic markers for breast cancer.
The effect of retinoids is controlled by nuclear retinoid receptors (proteins which bind to retinoids and change the production of other proteins). Recently we have found that retinoic acid receptor b, a nuclear retinoid receptor, is the only retinoid receptor whose expression is induced by retinoic acid in hormone-dependent, but not in hormone-independent cells, which correlates well with the observed retinoic acid growth inhibitory effect. Preliminary studies have further shown that the introduction of retinoic acid receptor b into a hormone-independent breast cancer cell line that lacks the production of retinoic acid receptor b restores the retinoic acid growth inhibitory effect. Based on these preliminary results, the proposed project will study the necessity of retinoic acid receptor b for retinoic acid growth inhibition in breast cancer cells. Efforts will be undertaken to study the retinoic acid receptor b production and the recovery of retinoic acid growth inhibition in different hormone-independent cells. Furthermore, retinoic acid receptor b selective retinoids will be used to specifically activate and counteract retinoic acid receptor b activity in both hormone-dependent cells and retinoic acid receptor b producing hormone-independent cells. Retinoic acid receptor b will also be eliminated from hormone-dependent cells using molecular biology techniques. Our preliminary studies have also demonstrated that cancer cell death can be induced by retinoic acid only in hormone-dependent cells. Therefore, the molecular mechanism by which retinoic acid receptor b induces cancer cell death and how the process contributes to the anti-cancer effect of retinoic acid will be studied.
A Peptide Vaccine for Breast Cancer Prevention
Esteban Celis, M.D., Ph.D.
Cytel Corporation
Breast cancer is a serious health problem both in the US and worldwide. In the US alone approximately 182,000 new cases of breast cancer are diagnosed each year and breast cancer is the most common malignancy and the third leading cause of cancer deaths in women (46,000/year). Because most of these deaths are the result of tumor recurrences and metastatic disease, new approaches to prevent disease progression such as immune-based vaccines must be seriously considered. The purpose of this project is to define the capacity of synthetic protein fragments (peptides) to induce cytotoxic T lymphocyte (CTL) responses to antigens expressed on breast tumor cells. Because CTL are the immune cells most capable of directly killing tumor cells, vaccines that induce these immune responses are an attractive way of preventing cancer recurrences or at least inhibiting tumor progression in women with early disease. Most significantly, such vaccines could be used to prevent the occurrence of breast carcinoma in healthy women who are genetically at high risk of developing this disease. This could be accomplished by immunization of young women from families with high rates of breast carcinoma.
CTL recognize antigenic peptides derived from processed proteins which bind to major histocompatibility gene complex (MHC) molecules. The MHC molecules, also known as the transplantation-rejection antigens are present in most cells including tumors and have the main function of presenting antigenic peptides to CTL. We aim to identify CTL antigenic peptides in a specific protein called HER2/neu, which is produced in high amounts in aggressive breast tumors. Potential CTL antigens from the HER2/neu protein have already been selected for these studies by the identification of those peptides from this protein that bind to the human MHC molecules. Following a protocol successfully utilized to identify melanoma tumor CTL antigens, we will test the MHC-binding peptides from the HER2/neu protein for breast cancer-specific CTL responses. These experiments will be done in tissue culture using blood lymphocytes from normal volunteers and breast cancer patients. The final goal of this work is to identify several peptides from the HER2/neu protein, which are capable of eliciting tumor-specific CTL. The outcome of these studies will be the basis of the development of these peptides into CTL-inducing vaccines used to prevent or delay progression of breast tumors that express the HER2/neu antigen.
Molecular Design for Prevention of Breast Cancer Progression
Joseph R. Couto, Ph.D.
Cancer Research Fund of Contra Costa
We propose to engineer a molecule that will be ideally suited for arresting metastatic progression of breast cancer. This molecule will be designed to deliver a destructive radioisotope only to breast tumor cells. Both single metastatic cells and large tumors will be efficiently targeted. When used in multiple periodic administrations, the molecule should be able to prevent the recurrence of metastatic breast cancer. Therefore, the success of this project should provide a means to reduce the mortality from breast cancer by prolonging or saving the lives of those afflicted with metastatic disease.
Our proposed IFab2 molecule will be related to “antibodies”, which are natural defense molecules that find their targets with exquisite specificity. Previous human clinical trials have demonstrated the ability of antibodies (attached to radioisotopes) to target metastatic breast carcinoma cells but they also point to some problems: (A), the large size of antibodies leads to suboptimal targeting rates and to poor penetration of large tumors, and (B), antibodies can stick to healthy defense cells. This sticking is desirable in the natural situation as a means to summon defense cells to destroy foreign invaders tagged by antibodies. In the clinical setting however, this can result in the destruction of healthy cells, in diminishing the rate of delivery of the radioisotope to tumor cells, and in other undesirable side effects. The proposed IFab2s will find their targets like antibodies but will diffuse more rapidly to metastatic sites. IFab2s will also lack the regions that bind healthy defense cells while retaining the high affinity and specificity for breast tumor cells.
Ifab2s do not occur naturally and cannot be prepared directly from antibodies. Essentially they will look like two connected and inverted antibody fragments. We propose to optimize their design, which will be encoded by a single engineered gene. This gene will direct the production of IFab2s for breast cancer treatment in unlimited quantities and ready for attachment to a radioisotope. The first IFab2s will have the targeting properties of our best humanized antibodies, BrE-3 and Mc3, which have been proven to target and specifically destroy human breast carcinomas. If successfully designed, these IFab2s could be used to completely arrest metastatic progression with negligible damage to non-tumor tissues.
Peptide Vaccines for Immunoprevention of Breast Cancer
Thomas J. Kipps, M.D., Ph.D.
University of California, San Diego
This project will examine how the immune system can be used to prevent recurrent breast cancer. About a third of all women with breast cancer have breast tumor cells that make a large amount of a particular protein, called erbB-2. Because of this, these tumor cells can be distinguished immunologically from normal cells. We aim to take advantage of this distinctive feature of breast cancer cells to develop a novel strategy to treat those patients who have tumors that make large amounts of the erbB-2 protein. Our research will investigate ways to induce an immune response against this protein in such patients. Such an immune response should destroy tumor cells that make erbB-2, and thereby protect such patients from developing recurrent breast cancer.
We propose to study synthetic peptide vaccines that can stimulate an immune response against cells that overexpress erbB-2. Because erbB-2 is a self protein, it ordinarily does not stimulate an immune response. However, synthetic peptides that mimic portions of the erbB-2 protein can induce an immune response against erbB-2, particularly when such peptides are physically coupled to an immunogenic protein or presented by activated immune cells that can initiate the immune response. The immune response induced by these synthetic peptides can result in the production of antibodies that bind erbB-2. Alternatively, they may activate “helper T cells” that can induce cytotoxic T cells and/or macrophages that specifically can kill tumor cells. We have identified candidate synthetic peptides that can induce such responses and have developed methods for identifying additional synthetic peptides for use as vaccines against recurrent breast cancer.
We plan to (1) characterize the antibodies induced by synthetic peptides of erbB-2 for their ability to affect adversely the growth or survival of breast tumor cells; (2) identify synthetic peptides of erbB-2 that can be presented to, and recognized by helper T cells; and (3) examine peptide vaccines for their ability to prevent breast cancer recurrence. Through these studies we may discover effective strategies for the active immune prevention of recurrent breast cancer.
Blocking Breast Cancer Progression with Radioactive Antibody
Michael Samoszuk, M.D.
University of California, Irvine
Approximately 20% of human breast cancers are extensively infiltrated by a rare type of white blood cell called an eosinophil and contain extracellular deposits of an enzyme called eosinophil peroxidase (EPO). Coincidentally, EPO has significant structural similarity to lactoperoxidase, an enzyme that is naturally found in breast milk and that is secreted by lactating breast cells but not by the resting breast cells. We have developed a monoclonal antibody called EOS that binds to EPO and possibly to human lactoperoxidase but not to any other normal human tissues. Based on pilot study data, we hypothesize that EPO and/or lactoperoxidase are present at high levels within small nests of breast cancer in certain patients. Consequently, we propose that EOS antibody will be an ideal vehicle for specifically delivering radioactive materials to the EPO/Lactoperoxidase target within the nests of breast cancer. The radioactive material will then damage or destroy the cancer cells and blood vessels within the tumor, thereby preventing the progression of the cancer without causing significant systemic toxicity.
In order to test this hypothesis, we first intend to retrieve up to 300 archival specimens of benign breast conditions and breast cancers of different types. These tissued specimens will be tested for the presence of EPO and/or lactoperoxidase. We will then chemically attach EOS antibody to rhenium-188. This radioisotope was selected because it has a short half-life, does not accumulate in normal tissues, and can be effectively administered at low doses. Following the radio labeling, we will determine the physical properties of the radioactive antibody to determine if it will be suitable for human use. When completed, these preclinical feasibility studies will translate into a compelling rationale for initiating subsequent clinical trails of rhenium 188-EOS, a novel radiopharmaceutical that will have value for preventing disease progression in a subset of breast cancer patients whose tumors contain EPO deposits or lactoperoxidase.
The Role of Plant Estrogens in Reducing Breast Cancer Risk
Pamela L. Horn-Ross, Ph.D.
Northern California Cancer Center
This study will address two questions: (1) whether estrogens found in plant foods can reduce breast cancer risk, and (2) whether obesity increases breast cancer risk only when a woman's diet does not contain a sufficient amount of these plant estrogens.
The estrogens found in a number of plants are similar to the drug Tamoxifen. Women with breast cancer who take Tamoxifen are less likely to develop cancer in the other breast and Tamoxifen is currently being tested as a way to prevent the development of breast cancer in high risk women. Eating plant foods, such as beans and whole grains, which contain a substantial amount of weak estrogens, may be a natural and cost effective alternative way to reduce breast cancer risk. In postmenopausal women, those who are overweight have higher estrogen levels and are at greater risk of developing breast cancer. We will also test the hypothesis that overweight women who eat a lot of foods which are rich in plant estrogens may lower their increased risk of breast cancer since the weak plant estrogens they are consuming block their own body's strong estrogens from binding with estrogen receptors, thus, preventing breast cancer cells from growing. The lower breast cancer rates in Latina women despite their greater obesity may be partly due to their higher intake of plant estrogens from bean sources. If confirmed, this hypothesis could have important implications for reducing breast cancer risk through diet, particularly in White, African American, and Latina women among whom postmenopausal obesity is an important health concern. Our study will include White, African American, and Latina women, age 50-79, residing in two Bay Area counties. Three-hundred twenty-five women with breast cancer will be identified through the State's cancer database and compared to randomly chosen women without breast cancer. Women will be interviewed in their homes in either English or Spanish about their diet and other breast cancer risk factors; body measurements, including weight and height, will be taken.
The results of this study will provide additional understanding of the role that diet plays in the development of breast cancer and may have important implications for breast cancer prevention.
Determinants of Breast-Conserving Surgery and Survival
Cyllene R. Morris, D.V.M., M.P.V.M., Ph.D.
California Public Health Foundation
Primary therapy for breast cancer in women with localized disease has changed substantially in recent years. Breast'conserving surgery (BCS) with radiation therapy is an equally effective alternative to mastectomy, and was recommended for most women with stage I or II breast cancer by the 1990 National Institutes of Health Consensus Conference. (For this study, stage I or II is defined as localized tumors without lymph node involvement, or tumors up to 5 cm in diameter, with or without extension to axillary lymph node nodes on the same side.) Nevertheless, fewer than 50% of women who are eligible receive BCS in California, even though mastectomies may have profound emotional consequences and do not improve survival rates in women with small tumors. The goal of this study is to determine the reasons for the apparent under-utilization of BCS in California. A better understanding of this phenomenon may reduce the human and economic costs of breast cancer. In particular, the physical and psychological impact associated with unnecessary mastectomies represent a human cost that can be minimized by understanding which factors affect the choice of surgical treatment.
The objectives of the project are to: (1) determine statewide and regional trends of BCS utilization and radiation therapy, (2) determine the most significant predictors of choice of surgery for BCS-eligible women, and (3) compare the five-year survival experience of women receiving BCS and BCS-eligible women receiving mastectomies. Data from the California Cancer Registry (CCR), including all reported diagnoses of first primary breast cancer within the state from 1988 to 1995, will be used in the analysis. This study will be the first to address the issue of BCS utilization and survival on a statewide basis. The CCR operates the most complete cancer reporting system in California, and the CCR breast cancer database will be complemented and enhanced through linkages with several additional statewide files. These linkages will enable the analysis of a wide range of factors which may influence the choice of surgical treatment for primary breast cancer, including socioeconomic status, education, insurance coverage, and comorbidities. These factors have not yet been evaluated together with information on clinical factors, treatment, characteristics of the patient, hospital, and county of residence as predictors of surgery received and subsequent survival. The database generated during this study will contain the most comprehensive population-based information on breast cancer in California. Given the large number of breast cancer cases in the state, the proposed study will have enough power to reach a statistically sound conclusion. In addition, the substantial variation in race/ethnicity in California allows for in depth analysis of possible differences in patterns of care among minority women.
Understanding why BCS has been underutilized in California is an important step towards ensuring that women with breast cancer have an informed choice and receive optimal treatment.
Exercise, Ovarian Function, and Breast Cancer Prevention
Lisa S. Shames, Ph.D.
University of Southern California
There is substantial evidence to suggest that estrogens play a key role in the biological cause of breast cancer. We recently observed in a large population based study, that exercise, including moderate exercise, may significantly reduce a woman's risk of breast cancer. We hypothesize that the reduction in risk with exercise is due to altered ovarian function, specifically, more frequent anovulatory cycles (i.e., a menstrual cycle without release of an ovum) and/or reduced serum estrogen (and progesterone) levels in ovulatory cycles which likely contribute to a reduced exposure of breast tissue to estrogen and thus reduce the risk of breast cancer. Studies of highly trained athletes show that women who engage in exercise for close to an hour or more per day have a higher frequency of anovulation, lower levels of estradiol (E2-the most active form of estrogen) and in some cases a shortened luteal phase length with associated lower estradiol levels.
The purpose of this study is to investigate the relationship of exercise to ovarian function across a range of physical exercise activity that non-athletes may reasonably be expected to participate in (i.e., sedentary to less than an hour per day). Specifically we will aim to: (1) determine the relationship between physical exercise activity and frequency of anovulation, (2) determine pre- and post-ovulatory cycle lengths of women exercising at these various levels, and (3) measure serum hormone levels of E2 in these women. We plan to study 300 college students between the ages of 18 and 25 years, who have never borne children, over 2 complete menstrual cycles. A questionnaire consisting of demographic information, exercise, diet and medical history will be administered.
This study should contribute significantly to our understanding of the relationship between physical exercise activity and ovarian function. With this relationship better understood, it would be possible to provide readily understandable, practical advice to women on how to reduce their breast cancer risk through physical exercise activities.
Alteration of Dietary Fat to Reduce Breast Cancer Metastasis
Kent L. Erickson, Ph.D.
University of California, Davis
The overall goal of this project is to determine how altered dietary fat may reduce the growth and metastasis (the spread to other parts of the body) of human breast cancer. Despite advances in surgical techniques and the development of aggressive therapies for the treatment of primary breast cancer, most deaths are caused by the metastasis. Since less than 10% of women with breast cancer have full-blown metastasis in distant organs when their cancer is first detected, if methods were developed to reduce or block metastasis, great advances could be made in the prevention of disease progression.
Some studies of human populations and disease patterns have reported an association between breast cancer and dietary fat intake, while others have reported that no such association (“positive correlation”) existed. A major focus of this proposal is to enable us to understand how certain dietary fats reduce human breast cancer metastasis.
We, as well as others, have shown that when animals were fed diets with high concentrations of a specific fatty acid, linoleic acid, which is abundant in dietary oils derived from plants, that their breast tumor incidence increased and the time required for tumor appearance was greatly diminished when compared to animals fed low levels of linoleic acid. The main differences between the human studies and animal studies were that fat intake in the animal studies were controlled during the progression of the tumor whereas the human studies relied on food intake histories (which are limited in their accuracy) often taken after the tumors have started to develop. However, numerous studies with human cell lines indicate a role of dietary fat in the alteration of mammary tumor metastasis. Thus, we hypothesize that dietary fats, particularly those containing fish oils (n-3 fatty acids), may differentially alter primary breast tumor growth and metastasis. We have chosen to focus on two mechanisms that alter the production and responsiveness to a fatty acid based mediator, prostaglandin E2: blood vessel growth, and expression of enzymes associated with metastasis.
From this work we hope to determine how altering the amount and type of dietary fat may have a significant effect on the spread of breast cancer throughout the body, and thus provide information that could be used to decrease the mortality of women diagnosed with the disease.
Does Change in Emotional Expression Mediate Cancer Survival?
Janine E. Giese-Davis, Ph.D.
Stanford University
This proposal addresses the CBCRP priority of Prevention of Breast Cancer, Prevention of Disease Progression. The overarching goal of this project is to determine the specific therapeutic component linking participation in supportive/expressive group therapy to observed differences in disease progression in women with metastatic breast cancer. Past research with supportive/expressive group therapy has shown that survival was extended in metastatic breast cancer patients by 18 months, doubling the survival time of a matched control group. It is vital that we begin to understand the possible therapeutic mechanisms and the underlying related physiological mechanisms responsible for this surprising finding of increased survival. Therefore, I propose to determine if change in emotional expression, amount of talk-time, or emotional control are therapeutic mechanisms related to survival, immune and endocrine function, and well-being in metastatic breast cancer patients who participate in a year of this group therapy.
A long-standing hypothesis links both incidence and progression of cancer with lower levels of emotional expression, or higher levels of repressive or suppressive coping styles. This literature has guided the present hypothesis. In the proposed study, each participant has been videotaped as they take part in the supportive/expressive group therapy sessions. A sample of these videotapes will be used to code the specific emotions and the intensity of those emotions expressed for each of these women. The emotions expressed will be related to each woman's endocrine and immune functioning, their psychosocial well-being, and their survival. If it is found that change in emotional expression is important to enhanced physical or emotional well-being, this information could lead to more specific psycho-social interventions for breast cancer patients. The ability to provide such specific interventions would reduce the human and economic costs of breast cancer in California not only in terms of extended lives, but of quality of life.
Breast Cancer Chemotherapy: Does It Impair Brain Function?
Mary H. Wieneke, Ph.D., M.S.
California School of Professional Psychology
Complaints of impaired mental functioning following chemotherapy (“chemo brain”) have long been common among adult cancer patients, but they are poorly documented in the literature; such complaints have most often been attributed to depression or anxiety. Impaired cognitive functioning (thought, concentration, memory) may be an important treatment risk that paradoxically results, in part, from the outcomes of early detection and aggressive treatment. The long term impact of such phenomena on emotional and psychological functioning, quality of life, and employment should be considered in evaluating the human and economic costs of breast cancer.
This research will expand and more thoroughly address questions raised in a previous single-group study in which significant impairments were found in treated women's functioning for up to one year following completion of chemotherapy. Women who had received chemotherapy for early stage breast cancer showed deficits in several areas of mental functioning despite high-to-normal pre-cancer lQs: attention and concentration; memory, both visual and verbal; mental flexibility and speed of processing; visual function; and motor speed. For the planned research, standard cognitive tests will be used to evaluate brain-behavior relationships in 150 Bay Area women with breast cancer to: (1) assess the long-term effects on brain functioning of conventional chemotherapy and tamoxifen for early stage breast cancer, and (2) identify the contribution of psychological and emotional distress such as depression/anxiety, on mental functioning. Type and amount of chemotherapy, length of treatment, chemotherapy toxicity, and menopausal status will also be assessed for their effects on cognitive functioning. Three groups of women with early stage (I or II) breast cancer who receive: (1) adjuvant chemotherapy (with or without tamoxifen), (2) tam-oxifen only, or (3) no chemotherapy, will be com-pared at three points in time: shortly after diagnosis, and at approximately one and twelve months following chemotherapy completion. The value of the proposed research is that techniques to minimize or treat these effects could be developed once the specific cognitive effects are fully documented.
