Pathogenesis

Cancer arises through a long process, in which many changes in the structure and function of cells and tissues must occur. Interfering with any one, or group, of these may potentially arrest the process and prevent cancer from developing or progressing. Thus, increasing our knowledge of these many processes and steps (collectively called pathogenesis) is essential to the development of more effective prevention and treatment methods.

Twenty-six grants were awarded in this priority area. These include research aimed at identifying and understanding the action of genes involved in cell growth control, studies on the actions of hormones and hormone-blocking drugs, exploring what allows some cancer cells to leave the breast and form tumors in other parts of the body, and testing the effects of immune responses to breast cancer.

Several of the research projects are aimed at identifying and understanding the actions of genes which are associated with the development of breast cancer or the capacity of cancer cells to spread to distant sites. This includes using new technology to search for as yet unidentified genes involved in breast cancer and to study the effect of alterations in known genes at various stages of breast development. Other studies will explore the role of the BRCA1 protein, including how it is guided to different parts of a cell and how it is related to both development of the normal breast, and the development and progression of breast cancer. Finally, one study is exploring a new technique to interfere with genes important to tumor growth.

Another group of studies will increase our understanding of factors which allow cancer cells to grow uncontrollably and spread to sites outside the breast. One of the issues being studied is how normal growth-promoting mechanisms (such as known growth factors and hormones) go awry, resulting in uncontrolled growth. Similarly, the disruption of mechanisms which normally control growth (vitamin A, proteins called JNKs) or promote normal cell aging (telomeres) are being explored. Another issue is how certain proteins made by tumor cells act to allow tumor cells to leave the breast and to settle in other areas of the body.

Together these research projects will provide critical knowledge on normal breast cell activities, and the ways in which these are disrupted in breast cancer, with the ultimate goal of developing ways to restore normal function to, or interfere with abnormal function of, these cells.

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Research Program Awards


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Identification of New Candidate Breast Cancer Genes

Donna Albertson, Ph.D.
Lawrence Berkeley National Laboratory

The nature of a cancerous or pre-cancerous cell differs from that of a normal cell in ways that allow the cell to escape from normal growth controls and grow without restriction. Often it ispossible to associate changes in the genetic material of the cell with this change in behavior; in some cases the cell acquires extra copies of genes, and in others good copies of the genes are lost. These altered regions of the genetic material, or genome, can be recognized and the search for new cancer genes begins with the isolation of the changed region of DNA. The region, many times larger than a single gene, may contain tens to hundreds of genes, and scientists face the challenge of trying to find which of these genes has changed, leading to the development of a particular type of cancer. In order to do this, it is first necessary to identify which of the genes no longer function properly in the tumor cells. Currently, the best method to accomplish this task is not clear, and new approaches are needed to facilitate gene discovery. Therefore, in this proposal we will use a technique called "Fluorescent In Situ Hybridization" (FISH) to investigate whether or not a particular gene product is being produced in a normal or abnormal way in the tumor cells. Using FISH we will be able to see what is being produced within each cell. To use this technique, a "probe" is made that corresponds to the gene product. If the cell is making this gene product, then the probe sticks to the cell and the cell, now labeled with a fluorescent color, becomes visible in the microscope. By comparing how normal and tumor cells become labeled by the gene probe, we will be able to determine if the gene is turned on incorrectly in the tumor cells.

We will use FISH to look at the genes from a region on human chromosome 20 that is present in many extra copies in 30% of breast tumors. Studies have shown that in patients with tumors of this type, there is a greater likelihood of metastasis, and the patients have shorter disease-free survival times. Thus, the activity of extra copies of a gene or genes from this region appears to play a role in promoting metastasis and is an indicator of probable poorer outcome. Work in identifying the breast cancer gene(s) in this region has resulted in the isolation of DNA which spans this region of the genome and the identification of a number of candidate genes by biochemical methods. Identification of these breast cancer genes will be important in understanding the pathology of breast cancer and for devising strategies for patient treatment. The FISH technique we will use in the research laboratory may itself form the basis of future tests to classify breast tumors with respect to the activity of these and other genes and so aid in earlier detection and clinical management of disease progression.

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The Role of Bax Gene in Breast Cancer Pathogenesis

Helene Baribault, Ph.D.
The Burnham Institute

In the normal breast, cells divide, multiply, and then die. This normal process of cell death is termed "apoptosis." Breast cancer cells are cells that have bypassed this process and do not undergo apoptosis.

One key regulator of apoptosis is a protein produced by the Bax gene. It is known that, in approximately one third of breast cancers, this gene does not function normally and the protein is not produced. It is also known that these cancers are more aggressive than the two thirds of cancers that have normal Bax gene function, and have a poorer response to treatment.

In this study, we propose to use a new, powerful genetic technique to produce mice with abnormal Bax genes in the mammary glands. This will allow us to study the events leading to breast cancer throughout the development of the mammary gland. We will then use these mice to test therapies specifically for women with Bax-deficient breast cancer. These studies will result in potential new therapies for breast cancer, and in new methods to study the effects of other breast cancer-related genes.

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Non-Genomic Actions of Antiestrogens

Myles Cabot, Ph.D.
John Wayne Cancer Institute

In many cases, breast cancers are stimulated to grow by estrogen, the female hormone. It is therefore beneficial to limit the amount of estrogen in contact with the tumor. One way to achieve this, without shutting down the body's production of this essential hormone, is to block estrogen from interacting with the tumor. This can be accomplished with chemicals termed antiestrogens, such as tamoxifen. Tamoxifen's molecular structure is similar to that of estrogen such that it binds to the estrogen receptor on the tumor, blocking the tumor from estrogen and not producing the stimulation of tumor growth that estrogen does.

Whereas tamoxifen is taken by millions of cancer patients world-wide, and is presently being investigated as a preventive agent in 16,000 healthy women, its popularity does not come without concern. Recent research has indicated that the drug increases chances of developing uterine cancer. Although tamoxifen is a potent weapon when used early in cancer treatment, officials at the National Cancer Institute warn against prolonged use. This warning was further underscored by the State of California's Proposition 65 to classify tamoxifen as an agent known to cause cancer.

It is now thought that some actions of tamoxifen occur through mechanisms as yet unknown, other than through blockade of estrogen receptors. We have discovered that tamoxifen can interact with the surfaces of cells to send signals inside the cell. From our initial experiments, we believe that these cell surface signal properties are key both to some of the more recently-discovered benefits of tamoxifen, such as reversing drug-resistant cancers to drug-susceptible cancers and to some of the adverse effects. We intend to elucidate the mechanisms underlying some of the adverse and beneficial effects of tamoxifen and, with this knowledge, develop new drugs to bring to the clinic.

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Degradation of Growth Factor Receptors and Breast Cancer

Gordon Gill, M.D.
University of California, San Diego

The growth of normal breast cells is tightly controlled growth by synchronized messages that promote growth and messages or processes which discourage growth. The messages which promote growth come from outside the cell in the form of protein growth hormones such as the one called EGF. EGF binds to the surface of cells by attaching to "EGF Receptor" molecules called "EGFR." The complex of hormone and receptor is then carried inside the cell where the two molecules are separated and a decision is made regarding whether the EGFR will be degraded or degraded to the cell surface.

The messages or processes which discourage or prohibit growth are less well known. However, progress has been made in this lab with the discovery of a group of proteins called the "sorting nexins" (SNX). These proteins appear to be responsible for the decision regarding whether the EGFR molecule is degraded or recycled to the cell surface.

Breast cancer cells have an excessive concentration of EGFR on their surfaces. It is not known why this is so or how it affects the dysfunctional growth which occurs in breast cancer; however, the SNX are likely to be involved. The excessive EGFR can bind an excessive number of molecules of the growth factor EGF. It may be that this excessive dose of growth factor is critical to tumor growth.

This project will: (1) analyze the structure of the portions of SNX which are responsible for its function in sorting the EGFR molecules to degradation or recycling; (2) isolate and evaluate the SNX molecule for sorting activity for other growth factors; (3) test whether increased SNX will inhibit the growth of breast cancer cells; (4) study how SNX functions in the cells by learning in which pathways it participates.

These studies address the pathogenesis priority of the BCRP. In addition, the SNX functional pathway may provide a very effective point for intervention in breast cancer tumor growth.

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Predictors of Recurrent Breast Tumors in Women with DCIS

Karla Kerlikowske, M.D.
University of California, San Francisco

The purpose of this research is to better define which breast ductal carcinoma in situs (DCIS) will recur after surgery. DCIS is a premalignant breast lesion confined within the mammary ducts that is detected primarily by mammography. Prior to the widespread practice of mammographic screening for breast cancer, DCIS occurred at a rate equal to 1-2% of breast cancers diagnosed in the United States. However, DCIS now accounts for a number equal to over 12% of all newly diagnosed breast cancers. Some DCIS lesions will go on to develop into invasive breast cancer. In addition, some DCIS lesions that are removed by surgery recur. At present, we cannot distinguish which DCIS lesions have the potential to develop into invasive cancer from those that never will. We also cannot distinguish which DCIS lesions will recur after surgery from those that never will. Thus, there is not consensus among doctors about the best way to treat DCIS. Some doctors recommend mastectomy (removal of the entire breast), while others recommend lumpectomy (removal of the DCIS lesion and the area of breast surrounding the lesion) followed by radiation therapy, and others recommend lumpectomy alone. Until recently, most women with DCIS have been treated with mastectomy. However, since it is recommended that most women with early stage invasive cancer undergo lumpectomy, the routine use of mastectomy for patients with DCIS has been called into question.

We are specifically targeting women with DCIS who were treated with lumpectomy alone since we are interested in identifying prognostic (predictive) factors that may lead to more individually appropriate recommendations for treatment. Since very few women receive no surgical treatment for DCIS, studying untreated women is not feasible. Specifically, we propose to conduct a population-based, nested case-control study (i.e., a study which compares women who had DCIS which recurred -- cases -- to a similar group of women without a recurrence -- controls -- drawn from a defined population of DCIS cases) to measure factors that may influence whether DCIS lesions recur as either DCIS or invasive cancer following lumpectomy. The population base is the nine-county San Francisco Bay area.

We will measure several different types of factors, including: 1) epidemiologic and clinical factors including information such as the age of the woman when DCIS was first detected, how the DCIS lesion was first discovered, whether the woman had breast symptoms when DCIS was first discovered, and whether there is a family history of breast cancer, 2) tumor factors such as size of tumor and morphology (i.e., the form and structure) of the premalignant cells; and 3) molecular markers of tumor function including estrogen and progesterone receptors, proteins uniquely made by tumor cells (p53 and erbB-2) and Ki67 which measures how fast tumors grow. A "cohort" (i.e., women diagnosed with DCIS during the period 1983 to 1992, who were over 40 years of age, and were treated by lumpectomy alone) of approximately 935 women will be identified from nine San Francisco Bay Area counties through the regional SEER cancer registry and interviewed by telephone to obtain information on epidemiologic and clinical factors. From these, all recurrent cases -- either of DCIS or invasive cancer -- (estimated to be 140 of the 935 women) and two controls (women without recurrent disease) per case (or 280 controls) will be selected from this cohort for inclusion in the study. For the 420 women selected for the nested case-control study, paraffin embedded tissue blocks (initial and recurrent blocks) will be collected by the regional tumor registry for standardized pathology review and determination of molecular markers.

This will be the largest study to date to investigate women with DCIS who have breast tumors recur and the only one to look at molecular markers in a population-based sample. The results of this study will be used to make recommendations on prognostic factors useful in predicting the risk of recurrent breast tumors after lumpectomy. Such knowledge will be useful to patients and clinicians since it will provide a better basis for determining appropriate treatment, such that those women who have a low risk of disease recurrence may avoid receiving unnecessary radiation therapy and those that have a high risk of recurrence may consider radiation therapy in addition to lumpectomy or possibly mastectomy.

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Abnormal Regulation in Breast Cancer Development/Metastasis

Ulla Knaus, Ph.D.
The Scripps Research Institute

Breast tumors evolve when a cell acquires multiple alterations in its genetic material, helping the cell to escape from normal control of its growth or movement. These changes give this cell a growth advantage. At some point in tumor development, selected cells can break away and travel through the circulatory or lymphatic systems, invade a distant organ and form a secondary tumor. This spread of malignant cells is called metastasis.

Invasion of tissue and metastasis are active processes which seem to be regulated by proteins that operate at the cell surface and inside the cell, constantly communicating with each other. Messages from the blood stream and surrounding tissue are given to individual cells and are transmitted to the inside of the cells and passed onto other proteins. These proteins interact with the target protein by modifying its activity or location. Important parts of this regulation involve enzymes known as protein kinases, which can activate or inhibit other proteins by attaching a phosphate group. The growth and formation of cancers depend on at least one such kinase pathway known as MAP kinase.

An additional communication pathway that may play a critical role in tumor development and metastasis involves a protein kinase termed PAK. PAK is tightly regulated in normal cells and seems to be directly involved in growth control, cellular shape changes and movement or motility responses, an important prerequisite for metastasis. How PAK regulates these cellular functions is not known. Identification of immediate partner proteins of PAK and analysis of this interaction should increase our understanding of cellular functions under normal control. We will then determine if these pathways are regulated in an abnormal fashion in breast cancer cells. PAK deregulation might lead to an increase in cellular motility, a hypothesis which will be investigated. Finally, we will study how PAK might be regulated by abnormal mechanisms in breast cancers.

The knowledge we obtain will lead to a greater understanding of the development of breast cancer, and will enable control of tumor progression by innovative treatments which reduce or prevent such spreading. Since cancers of the breast can be readily removed prior to their spread into additional organs, the studies described here form the necessary initial steps to understand the molecular basis of breast cancer, ultimately and directly leading to a greater ability to intervene effectively in this disease.

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Oncogene Regulation in Mammary Cancer

Robert Oshima, Ph.D.
The Burnham Institute

Breast cancer, like other types of cancer, is a genetic disease caused by the permanent changes of specific genes which result in unregulated growth of mammary gland cells. Progress in cancer and general biological research has identified a relatively large number of different genes which, when changed, can lead to the inappropriate growth of particular tissues. With the accumulation of one or multiple changes this abnormal growth develops into cancer. One of the most important discoveries of the last ten years has been the realization that many of these key individual genes act in different ways to transmit information from outside the cell through a chain of gene products and finally to the central nucleus where all the genetic information is stored. In the nucleus, these signals act through proteins called transcription factors to alter how much of our genetic information is read. In turn, the reading or transcription of multiple genes leads to the uncontrolled growth of cells and alterations in their behavior. The concept of a chain of different gene products which pass information to the nucleus has helped us to group and order the action of different gene products into pathways which act like a chain of command which passes information from one link in the pathway to the next. An inappropriate message to grow could be started at many points in the signal pathway. The main idea of this proposal is to try to correct at the nucleus the inappropriate signals to grow instead of at the many different points upstream which may start the signal. The multiple different signals which the cell receives seem to funnel through a limited number of key nuclear proteins in the nucleus. We will inhibit the action of one of the these key proteins, the Ets transcription factors, by engineering a mouse which expresses an inhibitory form of Ets specifically in mammary glands. This mouse will then be mated to another type of mouse which always develops mammary tumors due to the growth signals started by a mutant protein at the cell surface. The effect of inhibiting the Ets transcription factors on the appearance and growth of the tumors can then be evaluated. This work may identify a key target for controlling the growth of breast cancer cells. If the Ets transcription factors are essential for the unregulated growth of breast cancer cells, it may be possible to design drugs which interfere with the action of these specific proteins. The advantage of identifying a nuclear target for intervention is that in theory, such a target may be effective for many different tumors that are started because of a variety of possible upstream alterations.

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Immune Responses to Breast Cancers: Function of TRAF Proteins

John Reed, Ph.D.
The Burnham Institute

It has been estimated that potentially cancerous cells arise in our bodies nearly every day. Fortunately, however, cells within our immune systems are able, in most cases, to eradicate these abnormal cells before they have an opportunity to form lethal tumors. For over 100,000 women each year in this country alone, the immune defense mechanisms against tumors fail where carcinomas of the breast are concerned.

The goal of this proposal is to provide new insights into some of the mechanisms by which immune cells can attack and kill cancerous cells. Most immune cells attack tumors by producing proteins that bind to receptors on the target cancer cells. These receptors, in turn, deliver signals into tumor cells which activate a latent program for cell suicide. Understanding more about the mechanisms by which these suicide receptors function has the potential to provide novel insights that might be exploited clinically to enhance immune responses to breast cancers.

Some of the most important suicide receptors are members of the Tumor Necrosis Factor (TNF) Receptor family. Little is known, however, about how these receptors deliver signals into tumors that kill them. Recently, we and others have discovered a group of closely related proteins that interact with TNF-family receptors, called TNF-Receptor Associated Factor (TRAF) proteins. The purpose of this proposal is to understand the mechanisms by which these TRAF proteins transfer signals from the suicide receptors into breast cancer cells. The results of these investigations may provide mechanistic insights that lead to novel therapeutic approaches to breast cancer treatment and prevention.

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Estrogen Receptor-Interacting Proteins in Breast Cancer

Michael Stallcup, Ph.D.
University of Southern California

The normal development and function of the breast is regulated by a number of hormones including estrogen. Among other effects, estrogen stimulates many cells of the breast tissue to proliferate, i.e. to replicate themselves. Many breast cancer cells also require estrogen to stimulate their growth, and for this reason, drugs that block the action of estrogen are often used effectively in therapy of breast cancers. However, some breast cancers do not respond to these anti-estrogen therapies and, in addition, many breast cancers that initially respond to anti-estrogen therapy eventually become resistant. For these reasons, it is important to continue seeking new methods of treatment. The ability to design new treatments will depend upon our ability to understand the nature of cancer cells and how their growth is stimulated and regulated. Because estrogen remains a central factor in stimulating breast cancer cell growth, we are endeavoring to gain a more detailed understanding of the mechanism of estrogen action, since this knowledge will be essential for designing new therapeutic strategies to block estrogen stimulation of breast cancer cell growth.

Estrogen stimulates breast cancer cell growth by activating specific genes in breast cells that are responsible for cell growth and division. Much is known about this mechanism already. The hormone estrogen enters the cell and binds tightly to a specific protein, the estrogen receptor. The binding of the hormone activates the receptor, which then binds to specific genes in the cell nucleus and activates those genes. Some of these activated genes are responsible for stimulating cell growth. Why and how the binding of the estrogen receptor to specific genes activates those genes is still not known, and a better understanding of this phenomenon is the major goal of this proposed investigation. In order for the estrogen receptor to activate the genes that it binds to, it is believed to require assistance from other, as yet unknown, proteins in the cell. In our preliminary work, we have identified several novel proteins that interact with the estrogen receptor in a very specific manner: these novel proteins bind to the receptor only after the receptor has been activated by hormone; furthermore, they appear to interact with a specific portion of the receptor that is known to be important for the receptor's ability to activate the target genes. It is our hypothesis that these proteins play important roles in the action of the estrogen receptor. We therefore propose to identify these proteins and to determine the nature of their roles in assisting the estrogen receptor to activate specific genes in breast cancer cells.

These novel proteins may represent novel targets for breast cancer therapy, since blocking their action may prevent the growth of some tumors that do not respond to antiestrogen therapy. Furthermore, combination therapy, involving anti-estrogens and agents that block the action of these novel proteins, may prevent the recurrence of some breast tumors. More basic knowledge of how estrogen activates genes is essential before such novel therapies can be designed, and such knowledge is the goal of this proposal.

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How are Collagenases Involved in Breast Cancer Metastasis?

Alex Strongin, Ph.D.
La Jolla Institute for Experimental Medicine

Metastasis, the development of a breast cancer and the colonization of other organs by the tumor, requires several key destructive events, including the movement of tumor cells through tissues (invasion) and the movement of cells into and out of the blood stream. During these events, the tissue structures of the breast are changed by the tumor cells, allowing the tumor cells to move through the tissue and into blood vessels to establish metastatic tumors. Key players in this process are proteins called metalloproteinases (MMPs) and, in particular, the subgroup of collagenases, balanced with their protein inhibitors, which are abbreviated TIMPs. The MMP proteins are enzymes capable of digesting normal tissue proteins such as collagen. Collagen is essential to maintaining normal tissue structure. During fetal development, and in wound healing in adults, these two classes of proteins work together to carefully regulate tissue development or repair. Tumors, however, subvert the normal control mechanisms and turn on collagenases to digest the normal tissue collagen and thereby facilitate tumor invasion and metastasis. By understanding how MMPs and their inhibitors work and are regulated, we may learn how to control these proteins and arrest tumor growth and metastasis.

Recently, we have also identified a previously unknown cell receptor, called MTMMP, which seems to play a central role in regulating MMP collagenase activity. MTMMP sits on the surface of normal and tumor cells. Itself an enzyme, it appears to activate the tumor MMP proteins. Normal cells have MTMMP in an inactive, or switched off, mode but breast tumor cells may have the receptor always turned on or activated. The studies we will carry out will use recombinant molecular biology and biochemical methods to identify exactly how the tumor triggers the action of the tumor cell receptor MTMMP and the MMP collagenases and what molecules might be designed to block this mechanism of breast tumor cell invasion and metastasis.

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Innovative, Developmental and Exploratory Awards (IDEA)


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Altering Telomerase to Prevent Breast Cancer Progression

Elizabeth Blackburn, Ph.D.
University of California, San Francisco

The goal of this proposal is to explore two new strategies for blocking breast cancer progression and tumor maintenance. Both involve altering the action of telomerase in breast cancer cells, but in different ways. These are early experiments designed for the ultimate long term goal of using these novel strategies clinically, in the prevention and/or treatment of breast cancer.

Several years ago in my laboratory, we discovered the enzyme telomerase, which is the enzyme responsible for creating the DNA which makes up telomeres. Telomeres consist of DNA sequences, repeated over and over at the ends of chromosomes, which bind protein factors and make a "cap" securing the end of every chromosome, preventing it from fraying away. The telomerase enzyme adds short stretches of DNA to the ends (the telomeres) of all chromosomes as cells are dividing. This extra DNA makes up for the loss of DNA from each chromosomal end that is incurred by the natural inability of chromosomal DNA replication to replicate the chromosomal ends. Without replenishments by telomerase, as cells repeatedly divide, the telomeres gradually shrink. When telomeres become too short, cells stop dividing. Thus, for a cell to keep dividing, telomerase is needed in sufficient amounts to keep the chromosomes "topped up". Cancer cells, which divide much more than many normal cells in the body, often activate telomerase. This is thought to contribute to their ability to keep dividing.

We have discovered that telomerase is active in breast cancer cell lines grown in the laboratory. Therefore, we have decided to alter the action of telomerase in breast cancer cells in two very different ways, both designed to stop these tumor cells from dividing.

In the first strategy, inhibitors of telomerase will be used to prevent telomerase from maintaining telomeres. We have made the novel finding that some of the same kinds of drugs that stop the HIV-1 virus from replicating can also inhibit human telomerase, and cause telomere shortening in at least some types of human cells grown in the laboratory. Therefore, the first goal of this proposal is to test whether we can shorten telomeres in human breast cancer cells, using breast cancer cells explanted directly from patient biopsies into short-term primary cultures in the laboratory. Based on previous results with simple organisms, we predict that without telomerase working properly, the telomeres will run down until the cells stop dividing. In this way, we will find out about the role of telomerase in breast cancer progression and tumor maintenance. The ultimate long term goal is to determine whether telomerase inhibitors will be useful in the prevention and/or treatment of breast cancer.

The second strategy, rather than seeking to shut off telomerase action as the first strategy does, instead actually turns the action of an active telomerase against the breast cancer cells. This active telomerase is one we will have engineered to direct the synthesis of what we term "toxic" telomeric DNA sequences in breast cancer cells. When we do this in simpler organisms, they very quickly cease dividing because of these adverse telomeric sequences.

The planned research explores new strategies for potential treatment of breast cancer and will be greatly augmented by the multidisciplinary approach involving the combined expertise of this laboratory on telomeres and telomerases with those of Dr. Shanaz Dairkee in breast cancer cell explants and cell biology, and Dr. Robert Debs in gene transfer methods.

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Why do 70% of Breast Cancer Metastasize to Bone?

Jose Milla, Ph.D.
The Burnham Institute

One of the most important determinations that a physician must make when examining a patient afflicted by breast cancer is whether the tumor is confined to the breast or, instead, has already spread to other regions of the body. The process of cancer spreading is called metastasis, and once a cancer has progressed to the stage of metastatic disease, the probability that a patient will be treated successfully and cured are greatly reduced. The bone tissue is the preferred site of metastasis in patients with breast cancer.

In spite of the high frequency of bone metastases, the mechanisms that favor the skeleton as a preferred site for breast cancer spreading are completely unknown. The work proposed in this application aims at identifying molecules that may be providing this homing signal to the cancer cells. We will achieve this by using a molecular trick whereby billions of distinct protein sequences are examined for their ability to identify bone as a preferred target. We will identify and establish the building blocks of these small proteins and confirm that they are able to recognize bone specifically.

If this strategy is successful, subsequent experiments, beyond the scope of this proposal, will enable us to characterize the actual molecules present on the surface of breast cancer cells to understand, in detail, the mechanism of homing and metastasis to bone.

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Proper Relocation of a Tumor Suppressor in Breast Cancer

Marian Waterman, Ph.D.
University of California, Irvine

BRCA1 is the first breast cancer susceptibility gene to be identified as a tumor suppressor gene. The BRCA1 gene is either completely absent or mutated in over 80% of families with high rates of early-onset breast and ovarian cancer. While the identification of this gene is a major breakthrough in the study of breast cancer, a role for BRCA1 in sporadic breast tumors, which account for 95% of all breast cancer cases in Europe and North America, has been questionable since these tumors have normal BRCA1 genes. Recently, the BRCA1 protein was found to be completely missing from the nucleus in the majority of breast cancer biopsies examined and in most breast and ovarian cancer cell lines. The nucleus is a compartment of the cell where chromosomes are located and where genes important for cell growth and division are carefully regulated. Although the proper localization of BCRA1 is still under investigation, at least some BCRA1 protein is normally located in the nucleus. This finding, though preliminary, suggests that displacement of BRCA1 from the nucleus results from, or contributes to, the tumorigenic transformation of mammary cells, including sporadic tumors. Its role in this more common form of breast cancer should be reconsidered. Secondly, this observation suggests the possibility that forcing BRCA1 back into the nucleus of breast cancer cells might allow BRCA1 to carry out its normal tumor suppressor function and slow cell growth.

Proteins are guided into the cell nucleus in a multistep process that begins with binding of a nuclear import regulator to small nuclear localization signals within the protein. We have recently identified the genes for the two nuclear import factors known to bind such signals in proteins. Our goal is to use the genes for these import factors as tools to identify and study the nuclear transport pathway that normally guides BRCA1 to the nucleus, to search for a nuclear localization signal in BRCA1 protein, and to study its interaction with the nuclear import pathway in breast cancer cells. Since BRCA1 has only very recently been identified, little is known about the normal structure or function of the protein. Nothing is known about how it is normally transported to the nucleus.

Results from the experiments described here will be important to our understanding of the aberrant localization of BRCA1. It may also provide invaluable insight into the progression of breast cancer, since complete misplacement of BRCA1 is most common in end-stage breast cancer. Finally, our goal is to use the results from these experiments to design a modified BRCA1 protein that can readily move into the nucleus. We may then be able to judge whether forced relocalization of BRCA1 protein to the correct cellular compartment helps to restore normal growth control to these cells - a result that would have direct and important relevance in the search for new strategies to treat breast cancers.

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New Investigators Awards


Balance of Growth Factors in Breast Cancer Growth and Metastasis

Daisy DeLeon., Ph.D.
Loma Linda University

Many women die as a result of metastasis, the process by which tumor cells dissociate from the initial tumor and establish a new tumor at a different site. Currently, the most valuable predictor of breast tumor metastasis is the presence of cancer cells in lymph nodes. Nevertheless, new tumors reappear in 30% of patents with no cancer cells in the lymph nodes. Thus, more accurate prognostic indicators are needed. Our project is designed to investigate the basic biology of how three proteins interact (IGF-II, IGF-II receptor and cathepsin D) in breast cancer tumor growth and metastasis. Our proposal is relevant to the understanding of breast cancer development and metastasis, the major cause of death in breast cancer patients and an important BCRP priority area.

Cathepsin D appears to be a protein involved in metastasis. Several studies in breast cancer patients have demonstrated that high levels of cathepsin D correlated with significantly reduced cancer- free intervals after treatment. When studied by "immunocytochemistry" (the standard clinical laboratory assay) the correlation of cathepsin D and metastases have provided conflicting evidence. Thus, further studies with more precise assays for cathepsin D activity are necessary. Preliminary results suggest that high levels of "active" cathepsin D in breast tumors are associated with the risk of metastasis. The present study seeks to validate this observation.

IGF-II is a protein that stimulates cell growth and cathepsin D production. Both IGF-II and cathepsin D are secreted by breast cancer tumors and bind the IGF-II receptor. Despite distinct binding sites, IGF-II binding can affect cathepsin D binding to the IGF-II receptor. Thus, we believe that increased concentrations of IGF-II compete with cathepsin D for the IGF-II receptor; to restore the balance, increased secretion of cathepsin D occurs. We developed a breast cancer cell system that confirmed the theory and demonstrated how IGF-II blocked the transport of cathepsin D, increasing its secretion.

In summary, this proposal is designed to demonstrate that interactions between IGF-II and cathepsin D with the IGF-II receptors increase cathepsin D secretion, resulting in tumor growth and metastasis. To test this, we will determine whether: (1) IGF-II expression/cathepsin D secretion increases tumor growth and metastasis; and (2) Higher levels of specific forms of cathepsin D and IGF-II are found in breast tumor tissues than in normal breast tissues and are associated with metastasis. Expression of IGF-II and cathepsin D in paired normal and tumor tissue from breast cancer patients will be studied. Elucidation of the regulatory mechanism(s) in which these proteins participate and their interactions with factors contributng to the progression of breast cancer may have direct applications for improvements in patient treatment and follow-up.

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BRCA1 Regulation in Breast Cancer: A Rat Mammary Model

Donna Williams-Hill, Ph.D.
University of Southern California

Studies of Japanese atomic bomb survivors showed that the risk of developing breast cancer was highest among women who were irradiated at 0-9 years of age, while the risk of those exposed during puberty was significantly lower. Women who were over the age of forty at the time of the atomic blast were found to be at only minimally increased risk for breast cancer. More recent studies have found that inherited genes associated with the development of breast cancer, such as BRCA1, have been more frequently identified in young women (20-29) with breast cancer. Taken together, these observations suggest that the younger a female is when she is exposed to a breast cancer causing agent, the more likely she is to develop the disease. How or why this is is unknown.

Understanding the role of breast cancer associated genes during breast development may be key to designing more effective interventions and preventive strategies for women in all risk groups. However, dissecting such complex interrelationships in humans is extremely difficult for both practical and ethical reasons. One alternative is to utilize animal models to systematically analyze the factors associated with breast cancer risk, and mechanisms related to the cause of this disease. The rat mammary model provides a well characterized system for answering questions regarding how breast cancer develops in humans. The development of the mammary glands, and changes that occur in the breast during pregnancy, when mother rats nurse, and following weaning, are very similar to comparable events in humans, but occur over much shorter time frames. Using this model, we propose to study one of the genes thought to act as an inhibitor of tumors in the breast, BRCA1. Our studies will focus on how this gene acts in the mammary gland as it matures and during the development of breast cancer. The information gained will contribute to understanding whether there is a critical time in a female's life when breast cancer is most likely to begin, with the final goal of finding ways to prevent this disease.

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Postdoctoral Fellowship Awards


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Anti-Breast Cancer Activity of Vitamin A Derivative

Anissa Agadir, Ph.D.
The Burnham Institute

As the plague of breast cancer deaths continues, the need for new approaches to prevent this disease becomes increasingly apparent. With the perspective that cancer is a disease of uncontrolled cell growth, we need new agents to inhibit cancer cell growth. The family of molecules composed of vitamin A derivatives (retinoids) is an excellent set of candidates. Retinoids are currently used in the treatment of epithelial cancer and acute promyelocytic leukemia and have been evaluated as preventive and therapeutic agents for a variety of human cancers. Several studies show that retinoids are also capable of inhibiting breast cancer cell growth. However, their anti-breast cancer activity is mainly seen in early stage breast cancer cells and upon progression of the disease, breast cancer cells become refractory to retinoids. This phenomenon of retinoid resistance has been one of the major drawbacks in retinoid therapy. Unfortunately, how anti-cancer activity of retinoids is lost in late stage breast cancer cells is unclear. We propose to study the mechanism by which retinoids exert their anti-cancer activity in breast cancer cells and by which this effect is lost in resistant cancer cells. Results from these studies will provide us with an opportunity to increase retinoid sensitivity in retinoid-resistant breast cancer cells, and thereby enhance their therapeutic efficacy. The results will also provide a basis upon which to develop more effective anti-breast cancer retinoids with clinical value.

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Loss of Tumor Suppressor Proteins in Breast Cancer Cells

Sahn-Ho Kim, Ph.D.
Lawrence Berkeley National Laboratory

The breast contains several types of cells, one of which is epithelial cells. Breast epithelial cells grow and differentiate (to perform specialized functions) at precise times during embryological development, at puberty, and throughout adulthood. Breast epithelial cells are also the most common cells to give rise to breast cancer. That is, most breast tumors are composed of epithelial cells that fail to grow and differentiate normally.

Normal human cells, including breast epithelial cells, cannot divide indefinitely. The process which prevents normal cells from unlimited cell division is termed replicative or cellular senescence. Cellular senescence is a tumor suppressive mechanism. Indeed, most tumors develop their most malignant properties only after they have acquired enough mutations to overcome cellular senescence. Thus, breast epithelial cells that fail to senesce are very prone to develop into aggressive breast cancer.

The retinoblastoma susceptibility gene (Rb) is a cellular gene that is known to be critical for normal cellular senescence. Although it was first discovered as a tumor suppressor gene that was mutated in eye tumors (retinoblastoma), it is now known that Rb is mutated in many forms of cancer, including breast cancer. The Rb mutations that occur in cancer cells are those that delete or otherwise inactivate the Rb protein. Therefore, the normal Rb gene appears to be important in limiting cell division. Our preliminary data suggest that normal Rb limits cell division by binding to cellular proteins that are produced by senescent cells. However, genes that code for the cellular proteins that bind to Rb have not yet been isolated.

I propose to isolate the genes which are active in senescent human breast epithelial cells to code for proteins that interact with Rb. I will use a technique known as the yeast two-hybrid system, because this system has been very successful in isolating other interacting genes. I expect to identify genes expressed by senescent breast epithelial cells. These genes may themselves be previously unknown tumor suppressor genes. The isolation of these genes will provide us with critical information about how breast cancer is initiated, and how the abnormal growth of cancerous breast epithelial cells can be suppressed.

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Novel Ways to Control the Growth of Mammary Epithelial Cells

Susanne Koch, Ph.D.
The Scripps Research Institute

Each organ in our body is made up of cells which work together as a team to perform a specific function. To make an organ, the cells must increase in number (i.e., proliferate) and change their behavior (i.e., differentiate), acquiring the skills and teamwork necessary for proper tissue function. Cell proliferation and differentiation are controlled by special proteins, called growth factors or growth hormones. When you are cut, for example, growth factors are made which activate the growth and differentiation of new skin cells to heal the wound. When the wound heals, the growth factors are no longer made. Infrequently, this exquisite regulation breaks down when a single cell's genetic makeup gets changed by a process called "mutation." Consequently, this cell may alter its behavior and begin to proliferate in an uncontrolled manner. This process can lead to breast cancer when certain cells of the breast, the breast epithelial cells, grow uncontrollably. The growing mass of cells that stay in the breast forms a lump. Other cancer cells, especially those that are no longer differentiated and have "forgotten" they are breast cells, move away to form tumors elsewhere in the body. These undifferentiated cells are the most dangerous.

Growth factors bind to and activate proteins that sit on the cell's membrane. Activated growth factor receptors send signals into the cell to trigger cell proliferation and/or differentiation. These signals have to be tightly controlled to prevent unlimited cell proliferation. Growth signals can be stopped when the activated receptors and their bound growth factors are internalized and degraded. Our laboratory studies this process of receptor internalization, called "endocytosis." We have recently discovered that taking activated growth factor receptors into the cell can also change the messages they transmit, tipping the balance between triggering cell proliferation and differentiation.

The overall goal of my research is to understand the role of endocytosis in controlling the cell's response to growth factors. Conceivably, we can find a way to tip the balance toward differentiation and away from proliferation. To accomplish this, I propose to analyze how endocytosis affects the balance between differentiation and proliferative signals in breast epithelial cells engineered so that we can experimentally turn endocytosis on or off. Results from these studies will provide insight into novel ways to control breast cancer proliferation.

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Homeobox Genes: A New Class of Human Breast Oncogenes

Michael Lewis, Ph.D.
University of California, Santa Cruz

A genetic basis for cancer in general, and for breast cancer in particular, is now well established. Present evidence indicates that cancer initiation and progression is associated with alterations in multiple genetic elements, some of which may be inherited, while others reflect mutations occurring later in life. Candidates for breast cancer genes now include a large class of developmental regulators known as homeobox genes, based on our recent discovery that some of these are expressed in the development of the mouse mammary glad and are misexpressed (inappropriately turned on or off) in mouse mammary cancer relative to the normal mouse mammary gland. Homeobox genes normally function to establish cell identity and to provide positional information to the cell for proper patterning of the body and its organs as an animal grows from a fertilized egg to an adult.

Because homeobox genes are known to be critically important in the normal development of organisms ranging from flies to humans, and because cancer is a consequence of normal development gone awry, mutations in these genes may contribute to cancer. Development is the controlled acquisition of cellular identity (differentiation) and patterning (morphogenesis) over time. Cancer, in contrast, is characterized by the loss of cellular identity, patterning, and growth control. Therefore, a mutation in a gene that regulates these processes might cause cells to lose their differentiated state and their normal ability to form or maintain patterned structures, thereby contributing to cancer.

We propose to extend our studies of homeobox genes into the clinical arena of human breast development and cancer. We will study tissue samples from patients who have had breast tumors surgically removed; these studies will determine which homeobox genes are turned on and whether certain homeobox gene products are misexpressed in these tumors relative to adjacent normal human breast tissue. We will also determine which kinds of cells normally contain homeobox genes which are turned on, and in which kinds of cells the homeobox genes are normally turned off.

These studies, in conjunction with experiments we will conduct of mouse mammary cancer, will address the questions of whether these important developmental regulatory genes are normally functional in the human breast tissue and whether these genes show altered function in breast tumors. This work is designed to lay the foundation for determining whether or not homeobox genes contribute to the initiation and/or malignant progression of human breast cancer. At the clinical level, homeobox genes may eventually serve as targets for therapeutic agents, gene therapy or for markers for detection of tumor onset and tumor progression.

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Targeted Search for New Transcription Factors in Breast Cells

Claudia Lin, Ph.D.
Lawrence Berkeley National Laboratory

Breast cancer occurs as a result of alterations in the mechanisms that control normal cell behavior. Two of the major differences between a cancer cell and a normal cell are as follows: first, cancer cells acquire an inappropriate growth property; second, cancer cells generally lose their ability to serve the functions they normally have in a given tissue. These two aspects are intimately related in breast epithelial cells --the cell type from which more than 90% of breast cancers arise. In a normal breast cell, growth is stopped before the cell becomes functional (e.g., produces milk). This is controlled by changes in hormones in the female body and the network of large molecule proteins surrounding the cells. These changes can cause further modification in the type and quantity of regulatory proteins inside the cell, which in turn act as master swtichers for the growth and function of cells. Cancer cells, on the other hand, no longer respond to the environmental stimuli correctly, and do not make the proteins required for a controlled change in their growth and functional properties. The proposed work in this fellowship application will aim at identification and characterization of the regulatory proteins whose function may be lost in breast cancer.

We will use a cell culture system, developed in this laboratory, in which breast epithelial cells can be manipulated to either grow or become milk-producing cells. Using this system, we found that one class of regulatory proteins, called helix-loop-helix proteins (HLH for short), may be important coordinators of normal cell growth and function in the breast. We therefore set out to identify HLH proteins made in the breast cells and have identified one such regulatory protein. Since we know that the identified protein can only function when it is bound with another protein, we propose to search for proteins which interact with it. After we find the other protein(s), we will examine at what stages they are produced in our cells, and if their production is altered in human breast cancer cells. The proposed research will provide crucial insights into the molecular mechanism of breast cancer pathogenesis and provide useful information for early diagnosis and treatment of breast cancer.

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Why Does Normal Cell Death Not Occur in Breast Cancer?

Zheng-gang Liu, Ph.D.
University of California, San Diego

Recent progress in the understanding of breast cancer includes the finding that levels of a protein, called tumor necrosis factor (TNF) and its receptors (proteins which recognize TNF) are increased in the diseased breast tissue. The precise role of increased TNF production in the development of breast cancer, however, is unclear. It is also known that TNF is involved in cell death (which occurs in normal breast tissue, but fails to occur in breast cancer).

A critical change in the transformation from normal to cancerous tissue is the loss of control over cell growth. It has recently been learned that a different group of proteins, the JNKs, become active in the presence of TNF and are involved in the normal cell death process. Thus, studying the interaction between the JNKs and TNF will allow us to understand part of what has gone wrong to prevent normal cell death and allow the uncontrolled cell growth of cancer cells. It may also show us where in the process we can intervene to treat breast cancer.

The experiments in this proposal use a human breast cancer cell line to investigate the role of JNKs in TNF-induced cell death. Treatment with TNF induces these cancerous cells to undergo normal cell death. The project can be divided into two sections: 1) determining the role of JNKs in TNF-induced cell death; and 2) isolating the factors which affect JNK activity.

Because induction of normal cell death in cancer cells is one of the most promising ways to cure breast cancer, identification of components of this machinery will be fundamental for successful and rational breast cancer therapy.

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Zinc Finger Proteins to Study Breast Cancer Angiogenesis

Qiang Liu, Ph.D.
The Scripps Research Institute

Human breast tumors require the development of new blood vessels to supply nutrients for their growth. These newly established blood vessels can also serve as a channel for the tumor cells to spread. The process of blood vessel development initiated by tumors, called angiogenesis, has been clinically shown to be a major prognostic indicator for breast cancer progression. Proteins on the vascular cell membrane, called integrins, are necessary for blood vessel cell migration and invasion. This process of migration and invasion is in turn esential for the formation of the new blood vessels.

This project entails creating a new class of zinc finger proteins (finger-like modules that bind DNA and are stabilized by zinc ions) which are designed to inhibit the production of integrin proteins by binding to the integrin DNA. High affinity zinc finger proteins which bind specifically to the unique integrin DNA regions will be engineered and selected from zinc finger protein libraries. The zinc finger protein libraries are generated by inserting zinc finger protein coding sequences into M13 phages, a bacterial virus; the protein libraries are then displayed on the surfaces of the phages. This allows high affinity integrin DNA-binding zinc finger proteins to be selected through an in vitro binding and washing process. The evolved proteins will prevent the formation of new blood vessels in breast tumors by inhibiting their growth.

This provides a new class of molecules to study the process of angiogenesis in human breast cancer. Though this project targets the pathogenesis of breast cancer, the strategies developed here will find broad application to other cancers and to breast cancer therapy.

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Molecular Mechanisms of EGF Receptor Endocytosis

Alexandre Nesterov, Ph.D.
University of California, San Diego

A variety of proteins that are essential for normal growth and function of breast issue may cause cancer when inappropriately activated. Among them, the epidermal growth factor receptor (EGFR) is considered one of the main proteins elevated in breast cancer. A strong correlation has been found between the presence of high levels of EGFR in breast tumors and the aggressive potential of the tumor. Increased EGFR is implicated in metastasis, poor efficiency of tamoxifen therapy, and low survival rate.

EGFR molecules are located on the surface of cells that compose the mammary gland and, when activated by binding of their corresponding hormone, start a chain of events which leads to growth of these cells. It is not known how the normal balance of growth factor and its receptor is maintained or what is dysfunctonal in breast cancer. However, when too many EGFR are produced by the cell or too much hormone is available, excessive growth occurs, which contributes to the appearance of breast cancer.

Cells possess a mechanism to control EGFR related growth: when EGFR are activated, cells rapidly clear them from the surface and then destroy them. As soon as EGFR are activated (by binding the corresponding hormone), they are delivered into small "pits" on the cell surface. These pits then punch-off, form small vesicles and submerge into the cell interior. There EGFR are separated from the other contents of vesicle and delivered to a special apparatus that is responsible for digestion of cellular proteins. It is thought that special proteins are responsible for specific recognition of EGFR in the both steps of this process: EGFR clearance from the cell surface and their sorting to the degradation pathway.

One protein responsible for the sorting of EGFR to degradation pathway has been recently identified in our laboratory. However, proteins that are responsible for clearance of EGFR from the cell surface are still unknown.

The proposed research will pursue two specific aims: (1) identificaton of hypothesized, but currently unknown, components of the EGFR degradation machinery; and (2) targeting of these components for suppression of growth of breast cancer cells.

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Potential Tumor Suppressors in Breast Cancer

Mathias Treier, Ph.D.
University of California, San Diego

Epithelial cells in the breast grow and die in response to signals delivered by hormones including estrogen, retinoids (oxidized forms of vitamin A), thyroid hormone (T3) and progesetrone. Once they lose their ability to respond to these hormones they start to divide indefinitely, becoming breast cancer cells. For this reason, it is important to understand at the molecular level how these hormones exert their functions, thereby opening the possibility to interfere and regain control over breast cancer cells by administering hormone related drugs.

It is now generally accepted that hormones have to bind to a family of proteins, called nuclear hormone receptors, to exert their functions. Nuclear hormone receptors can bind to DNA and modulate the genetic program of the cell. Depending on the hormone they bind, they can either stimulate or inhibit the growth of cells. The underlying mechanisms for the different behavior of related nuclear hormone receptors are not well understood.

A step toward the understanding of this problem was achieved in our laboratory through the identification of a large protein named N-CoR (nuclear receptor co-repressor). This protein associates with nuclear receptors in the absence of hormones. Once these receptors bind their respective hormones, N-CoR loses its ability to bind to the nuclear receptors resulting in a change of the genetic program in the cell. Therefore, mutations in N-CoR which prevent the binding to nuclear receptors may affect cell growth control. Intriguingly, the effect of altered N-CoR function may be paradoxically opposite, dependent on the circumstances.

In this project we will study a possible role for N-CoR in breast cancer formation. As breast cancer is a multifactorial disease with risk factors relating to hormonal exposure, e.g. pregnancy, diet, environmental agents and heredity, this complex interplay is best studied in an in vivo situation. Mice are useful model systems to gain new molecular insights into breast cancer fomation. We will artificially create a situation in mice where N-CoR function is lost and vice versa where N-CoR protein is abundant. We can then directly assess the consequences for growth and survival of cells in the breast. In addition, breast cancer cell lines will be screened for altered N-CoR proteins. Identification of mutations in the N-CoR gene together with the understanding of its in vivo function would give us a new molecular tool to evaluate the aggressiveness of tumors in breast cancer.

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