Pathogenesis

The BCRP priority issue of Pathogenesis focuses on the mechanisms of the initial development and progression of breast cancer. The complexity and variety of projects that address this topic is a reflection of the amount of new and evolving scientific information on breast cancer cell and molecular biology, immunology, and human genetics. Fortunately, recent breast cancer pathogenesis-oriented research has opened many promising targets for early detection, prevention, diagnosis, and therapy. The efforts to translate many of the specific topics mentioned below into the treatment domain is seen in another BCRP funding priority area: Innovative Treatment Modalities. The potential for translation of basic science projects is seen as a key concern of breast cancer advocates.

First, the discovery of inherited breast cancer genes (e.g., BRCA 1 and 2) has sparked an increased effort on uncovering and understanding (Mark Chapman, PI) both inherited and acquired genetic risk factors. A primary focus of this work is defining the specific genetic differences between normal and cancerous breast cells. Two grants propose to identify novel breast cancer genes by either a fingerprinting technique (Sergei Malkhoysan, PI) or using a viral-based, peptide strategy (Manuel Perucho, PI). Unique breast cancer genes are a potential target for new, clinically focused detection methodologies (David Zarling, PI), and this project also examines novel genes believed to be associated with genetic recombination in breast cancer cells. Another project (Janis Jackson, PI) studies a gene showing a key mutation in many breast cancer patient samples. Specific genes and methods of gene regulation that affect cell growth/cell cycle are explored in two grants (Dieter Wolf, PI and Kevin Sato, PI). The application of novel gene technology has the potential of reversing or ‘reprogramming’ breast cancer cells (Steven Frisch, PI). Finally, specific DNA-associated proteins present in the nucleus of breast cancer cells may globally affect gene expression (Sanjeev Galande, PI).

A second approach to study breast cancer Pathogenesis targets the cellular interactions with the immediate environment and the mechanisms breast cancer cells use to spread in the body (metastasis). The extracellular matrix that surrounds cancer cells is thought to be able to regulate gene expression and influence cancer formation (Philippe Pujuguet, PI). The breast cancer cell surface receptors that bind to extracellular matrix proteins control metastasis, and two projects look at how cells regulate the ‘activity’ of these receptors (David Rose, PI and Emme Lin, PI). Both the metastatic spread and new blood vessel growth (angiogenesis) in cancers are appreciated as related processes. Cells can also degrade their immediate environment by specific enzymes, some of which may be unique to breast cancer and targets of future therapy (Pierre-Yves Desprez, PI). Breast epithelial cells interact dynamically with other supporting cells called fibroblasts, and this may become altered with cellular aging as a precursor to cancer (Judith Campisi, PI).

A third area of BCRP-funded research focus includes hormonal factors, growth factor receptors, and their associated intracellular signaling pathways. Ultimately these receptor-based signaling cascades of proteins affect gene expression, which underlie the differences between normal and cancerous breast cells. Funded projects will study a newly-discovered form of the estrogen receptor (Ruth Lupu, PI) and the loss of the progesterone receptor from breast cells (G. Shyamala, PI). Growth factor receptor regulation in breast cancer cells (Ichiro Maruyama, PI and Wanda Reynolds, PI) and the proteins that bind to them (ligands) (Fabiana Guerra-Vladusic, PI) are a focus of understanding breast cancer progression and new targeted modes of therapeutic intervention. Cell growth and death (apoptosis) pathways (Juan Zapata, PI and Heidi Theisen, PI) in the cell continue to be explored for specific functions in breast cancer biology. It is appreciated that the body ‘attacks’ breast cancer, but the cancer cells evade the controlling signals present in normal cells. Finally, the autoantibodies present in breast cancer patients offer a possibility of identifying the specific defective proteins of the cancer cell that are associated with pathogenesis (Robert Ochs, PI), and these could represent the best targets of therapy for individual women.

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Innovative, Developmental & Exploratory Awards – Type One

 

Does Cell Aging Cause Breast cancer?

Judith Campisi, Ph.D.
Ernest O. Lawrence Berkeley National Laboratory

The single largest risk for developing cancer is age. The exponential rise in cancer incidence with age has been explained by the accumulation of a critical number of mutations, primarily in the epithelial cells that give rise to the majority of malignant tumors in adults. However, it is unlikely that mutation accumulation completely accounts for cancer. Recently, epigenetic factors, such as the microenvironment, have become appreciated as contributors to cancer.

Little is known at the molecular level about the role of cellular aging in breast cancer. We propose that aging changes the breast epithelial cell microenvironment and contributes to cancer. Further, the age-dependent environmental changes may act in synergy with genetic mutations. We propose to study ‘replicative senescence’, which is the fixed number of cell divisions that a cell type is capable of in the body. This type of aging limits cell division by imposing an irreversible block to cell cycle progression. Interestingly, ‘replicative senescence’ also causes cells to become resistant to apoptotic (programmed) cell death. Recently, we provided the first evidence that senescent cells exist and accumulate with age. Our preliminary data suggest that senescent human fibroblasts express activities that can ‘relax’ the environmental control on breast epithelial cells. For example, senescent fibroblasts secrete extracellular matrix-degrading enzymes and epithelial cell growth factors. These factors could stimulate the growth and/or other malignant properties of neighboring breast epithelial cells.

We plan to study the role of senescent fibroblasts in mediating the transformation of breast epithelial cells in culture. Human fibroblasts from breast and non-breast origins will be co-cultured with murine and human breast epithelial cells having different potentials for growth, migration and invasion. We will use both monolayer-type (one cell layer thick) cultures and three-dimensional cultures containing various extracellular components. We will measure epithelial cell growth, differentiation, cell-cell and cell-extracellular matrix interactions, cell migration and invasiveness. Further, in other experiments we plan to relate senescence in fibroblasts to breast epithelial cells in which we insert either normal or mutated genes associated with breast cancer. We will explore the feasibility of inhibiting the "pro-carcinogenic" activities of senescent fibroblasts.

These experiments will provide a molecular framework for understanding the role of cellular aging in breast cancer. Future treatment of breast cancer may focus on counteracting these senescent changes in the microenvironment as an alternative to directly targeting the cancer cells.

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Novel Breast Cancer Epithelial Cell Metalloproteinase

Pierre-Yves Desprez, Ph.D.
California Pacific Medical Center

Breast cancer occurs as a result of alterations in the mechanisms that control normal cell behavior. Cancer and normal cells differ in two fundamental ways: (i) cancer cells grow inappropriately and (ii) cancer cells generally lose their ability to serve the normal functions. These two differences are closely associated with breast epithelial cells, the cell type from which more than 90% of the breast cancer arise. In addition, cancer cells may migrate (metastasize) from the breast and invade the lymph nodes and other foreign sites (eg., lung, bone). Our research will provide critical insights into how breast epithelial cells lose their proper control of growth and function and, in particular, how they acquire the ability to become invasive to initiate metastasis.

We will use a breast cell culture system developed previously to carry out these studies. Using this cell system, we have already shown that a specific intracellular protein (Id-1) is critical for coordinating the growth and function of the cells. When the Id-1 level is elevated inappropriately, it causes the cells to lose their normal functions and become invasive. Further, we have found that cells with an elevated level of Id-1 protein will secrete an extracellular enzyme (a metalloproteinase), which can potentially destroy the extracellular matrix surrounding the breast epithelial cells. Therefore, this enzyme may be a crucial factor in making the breast epithelial cells abnormally invasive in cell culture, and it may also contribute to the invasive potential of some aggressive breast tumors. We will study this metalloproteinase enzyme by isolating it using two different approaches. After isolation, we will have both the protein sequence and the DNA probes, which will then allow us to answer several fundamental questions. For example, how is the novel metallo-proteinase related to other described enzymes of different size and distribution? Where is this enzyme produced in the breast and other organs in the body? Is this enzyme overproduced in the breast of breast cancer patients? Our eventual objective is to determine whether this enzyme can trigger metastasis in breast cancer cells.

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Reprogramming Breast Cancer Epithelial Cells

Steven M. Frisch, Ph.D.
The Burnham Institute

Most breast cancers originate from previously normal breast epithelial cells. During this transformation into cancer cells, the epithelial cells often lose the expression of cell adhesion molecules. These transformed breast cancer cells are no longer regulated by normal cell-cell contact and environmental factors. Also accompanying this is the loss both of cell polarity and of a sensitivity to being detached from the extracellular matrix (anoikis-sensitivity), that in normal cells would cause cell death (apoptosis). We believe that at certain initiation (and perhaps advanced) stages of breast cancer there exist opportunities to reverse the cellular changes associated with malignant transformation. It is the focus of our research to identify cellular mechanisms to reprogram cancer cells back into their previously normal phenotype. In some respects this is analogous to reversing an epithelial-to-mesenchymal differentiation that occurs in development.

Recently, our laboratory has identified a viral gene product that converts human carcinoma cells and mesenchymal tumor cells into normal epithelial cells. This gene product, called the adenovirus ‘Ela’ 243 amino acid protein, is a nuclear DNA (gene)-regulatory protein. Ela does not bind directly to DNA, but acts as a cofactor with other DNA ‘adapter’ proteins, such as p300 and CBP. It is thought that the ‘signal’ for such DNA regulation relates to the critical adhesion events that are lost when cells become cancerous. Thus, Ela serves to selectively repress mesenchymal genes, thereby maintaining cells in the normal epithelial state. We think that the normal epithelial cell represents a default state. Moreover, it suggests that carcinoma cells and mesenchymal tumor cells can be induced to revert into this default state simply by inactivating the p300/CBP adapter proteins. The ‘default-phenotype’ hypothesis will be tested by determining if epithelial conversion can be achieved by inactivating the adapter proteins without the use of Ela. Our experiments on Ela and the target adapter proteins will use tumor cell lines and the primary method will be microinjection of antibodies and other compounds.

Our hypothesis of reprogramming transformed cancer cells with Ela opens several novel avenues to treatment. An added feature of this approach is that it could be useful even if only a fraction of the tumor cells are ‘reprogrammed’. Ela-converted cells become targets for immune response in the host, which can attack neighboring tumor cells. Further, the Ela protein could serve as a ‘template’ for designing drugs that mimic its activity.

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Analysis of Rac Mutations in Breast Cancer

Janis H. Jackson, M.D.
The Scripps Research Institute

The cellular changes involved in the transformation of normal cells to cancerous cells includes a category of regulatory proteins involved in a process termed signal transduction. These signaling proteins can ultimately determine the synthesis of specific genes necessary for tumor cell behavior, cell division, or other metabolic changes in cancer cells. These cancer-promoting proteins/genes are often referred to as oncogenes. They alter cell behavior when they are expressed inappropriately or become mutated. Recent studies have shown that the Rac 1 oncogene, a member of the Ras superfamily of proteins, may play an important role in carcinogenesis and/or metastasis. First, Rac 1 has been shown to (i) be required for Ras-induced malignant transformation, (ii) induce cell cycle progression and DNA synthesis in Swiss 3T3 fibroblasts, and (iii) transform normal cells into malignant cells. Second, Rac 1 protein is known to regulate an activity of phagocytes (NADPH oxidase) that is believed to contribute to the pathogenesis of many human tumors. Third, Rac 1 (in conjunction with Rho and/or CDC42) controls the assembly of polymerized actin structures (cytoskeleton) and associated cell surface adhesion complexes; and it is thought that these abilities enable Rac 1 to play a key role in regulating tumor cell motility, adhesion, and metastasis.

We hypothesized that Rac 1 might be mutated in human breast cancers, and be responsible for certain aspects of its pathogenesis. To initially test our hypothesis, we isolated RNA from metastatic, infiltrating ductal human breast carcinomas, and directly sequenced it (through PCR-amplified DNA) to detect the presence of Rac 1 mutations. Our preliminary results showed a mutation in codon #12 in 4 of the 5 breast carcinoma tissues. To our knowledge, this is the first demonstration of mutated Rac 1 in human cancers.

The overall goal of this IDEA research project is to confirm and expand on these findings to determine if Rac 1 is mutated in human breast carcinomas. If Rac 1 is found to be frequently mutated and activated in human breast cancers, it may provide a novel target in the development of anti-breast cancer therapeutics.

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The Role of a Novel Estrogen Receptor in Breast Cancer

Ruth Lupu, Ph.D.
Ernest O. Lawrence Berkeley National Laboratory

One of the major challenges facing breast cancer research today is developing and testing more effective chemoprevention systems, mainly through pharmacological means. Tamoxifen (Tam), a synthetic anti-estrogen, is widely used for the treatment of breast cancer. The administration of Tam to patients with node negative breast cancer induces substantial regression of the tumor and an increase in disease-free survival. The use of Tam was initially restricted to women with estrogen-receptor (ER)-positive tumors. For a number of years, it has been postulated that estrogen and anti-estrogens may have biological effects that are independent of the estrogen receptor.

About 50% of post-menopausal breast cancer patients who are ER-positive (ER+) benefit from anti-estrogen therapy. However, several aspects of this hormonal therapy are still puzzling. There are a large number of ER+ breast cancer patients who do not respond to anti-estrogens. Many patients who initially respond to anti-estrogen therapies eventually acquire anti-estrogen resistance, despite retention of the ER in many of the tumor cells. There are a large number of ER-negative patients who benefit from anti-estrogen treatments. Thus, the simple presence of the classical ER status is insufficient to recommend anti-estrogen treatment and the simple absence of the classical ER is not sufficient to recommend against anti-estrogen therapy. One current working hypothesis is that the expression of ER variants, which are incomplete ER molecules, can suppress the normal function of the ER. This hypothesis has yet to be proven.

Another exciting hypothesis has very recently emerged that may explain the apparently anomalous behavior of some breast cancer tumors with regards to their response to anti-estrogen treatments. A new ER coded by an entirely different gene than the classical ER, has recently been identified. This new ER is now termed ER-b. We have shown that this ER-b is expressed in normal breast and tumor tissue, and it is up-regulated after 48 hours of estrogen treatment. We hypothesize that this new ER-b is a player in suppression or induction of the normal ER-a function. The proposed studies should provide valuable information regarding the role of ER-b, help predict the tumor response to anti-estrogen therapy, and identify those patients that are likely to benefit from anti-estrogen treatment.

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Molecular Karyotyping of Breast Cancer by DNA Fingerprinting

Sergei R. Malkhosyan, Ph.D.
The Burnham Institute

The initiation and progression of human breast tumors involves a wide spectrum of known, suspected, and undiscovered genetic alterations. Thus, it is believed that tumor cells are genetically different from the surrounding normal breast cells. One potential approach to detect, analyze, and enumerate these genetic differences in breast cancer is to use a technique of DNA fingerprinting. We plan to use this technique to provide a comprehensive view of the role of known genetic aberrations in breast tumorigenesis and to facilitate the identification, mapping and eventual isolation of novel cancer genes. Our specific detection method is termed ‘Arbitrarily Primed PCR’ (AP-PCR). This is an unbiased method, which generates a profile of quantitative differences between the tumor and corresponding (from the same patient) normal tissues. We call it quantitative allelotype or ‘amplotype’. This ‘amplotype’ reflects tumor-specific changes of different DNA segments randomly distributed throughout the genome, and the chromosomal origin of the fingerprint bands can be easily determined. We have previously used this method to analyze colon cancer, where progression is closely associated with specific genetic defects.

Our experimental approach features three specific aims. First, about 100 human breast tumors with corresponding normal tissues will be analyzed by AP-PCR fingerprinting, which is anticipated to yield a ‘fingerprint’ of about 500 different DNA pieces (bands). Each difference between the matched tumor and normal sample will be linked to a chromosomal site and will be scored either as an allelic (genetic) loss, allelic gain, or structural change. Secondly, the extent (genetic damage index) and spectrum of genetic alterations will be determined for each tumor. Thus, we will compare breast tumors from different patients. Our goal is to associate these genetic aberrations with specific biological and clinical parameters. Finally, a sub-chromosomal map of recurrent genetic aberrations will be determined. These maps will then be compared with known genetic regions for breast cancer development. Our goal is to identify novel genetic aberrations for further study. Our experimental approach directly studies samples from breast cancer patients. It should provide a comprehensive view of the role of known genetic aberrations in breast tumorigenesis and facilitate the identification, mapping and eventual isolation of novel cancer genes.

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Growth Factor Receptor Activation in Breast Cancer

Ichiro Maruyama, Ph.D.
The Scripps Research Institute

Cell-surface receptors are proteins that bind factors such as hormones or growth factors, and transmit the external signal inside the cell, ultimately resulting in processes such as cell growth and differentiation. These receptors reside on the cell membrane and typically consist of three parts: a sensing segment that sticks outside the cell; a middle segment that runs through the cell membrane; and a segment that sticks inside the cell and transmits the signal inside the cell. Epidermal Growth Factor Receptor (EGFR) is such a cell surface receptor. It is present in a variety of different cell types, and is implicated in breast cancer. When EGFR is turned on (activated), it can enhance the mobility of cancer cells and increase their capacity to metastasize. Therefore, knowledge of a molecular mechanism for the receptor activation is a crucial step in the understanding of human breast cancer.

It believed that one way a receptor can be activated is by a hormone binding to two molecules of the receptor and bringing them together. However, an accumulating number of observations suggest that linking of two receptor molecules is not sufficient for the receptor activation. For example, the artificial linking of two receptor molecules by antibodies does not always activate the receptors. To clarify the controversial observations, we analyzed the structure of the membrane-spanning region of the aspartate receptor, a cell surface receptor for the amino acid aspartate. Based on the results, we have proposed a model for the activation mechanism in which the membrane-spanning segment rotates or twists in the plane of the cell membrane to transmit the factor-binding signal into the inside of the cell. We propose to determine whether this model also explains how other receptors, including EGFR, are activated. We will artificially mutate the membrane-spanning region of EGFR to find the structure of the segment critical for the signaling. We will also analyze the movement of the membrane-spanning segment during the signaling. Mutant receptors that can be activated without bound factors may also be isolated in this project.

Knowledge of a molecular mechanism for activation of EGFR should be a crucial step, not only in an understanding of the pathogenesis of human breast cancer, but also towards development of anti-cancer drugs. Using computer modeling, small chemicals or peptides that inhibit activation of EGFR can be designed based on knowledge of the molecular mechanism.

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Innovative, Developmental & Exploratory Awards – Type Two

 

Autoantibodies in Breast Cancer

Robert L. Ochs, Ph.D.
The Scripps Research Institute

Initiation and progression of breast cancer is believed to include the expression of novel and mutated cellular proteins. We plan to examine the blood of women with breast cancer for the presence of circulating autoantibodies to tumor proteins. Our initial studies revealed a wide spectrum of immunologic responses to specific protein antigens. Blood from 158 patients with breast cancer were initially screened using indirect immunofluorescence (a visual analysis giving cellular location) and immunoblotting (detects specific proteins) against breast cancer (T47D) cell and whole-cell extracts. Autoantibodies were detected in 78/158 of the sera samples. We are able to detect the location within the cell (e.g., nucleus, cytoplasm, mitochondria) where the autoantibodies bind. These autoantibodies may be targeting important proteins known to be involved in cancer, such as the p53 tumor suppressor protein, but most of them remain to be fully identified. Interestingly, a few patients had autoantibodies that recognized certain proliferation-associated proteins (NuMA and PCNA) not previously described in breast cancer. In summary, we believe that breast cancer may be rendering some molecules immunogenic and generating antibodies that, although not effective in combating the disease, might still be instructive in identifying novel proteins involved in malignancy.

In this project we will explore and further characterize the autoantibody specificities in the blood of women with breast cancer, and focus on comparing malignant to benign disease. The autoantibody specificities will continue to be determined by immunofluorescence microscopy and Western blotting. Once the autoantibody specificity of a breast cancer patient is characterized, we will then compare these to the clinical information to associate them with disease type, severity, prognosis, and outcome. Our long-term goal is to link information on autoantibody status (i) to achieve an earlier diagnosis, (ii) as a predictor of disease progression, and (iii) as a guide to select the appropriate treatment therapy. In addition to their direct clinical application, autoantibodies found in the blood of breast cancer patients could aid in the discovery of novel oncogenic proteins that are involved in the pathophysiology of breast cancer.

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Identifying Novel Breast Cancer Tumor Suppressor Genes

Manuel Perucho, Ph.D.
The Burnham Institute

It is widely accepted that breast cancer arises because of a loss of equilibrium between positive and negative regulators of cell growth. Genes with positive roles in cell growth are called oncogenes. When oncogenes are mutated or overexpressed they can act in dominant fashion to accelerate cell proliferation. In contrast, genes that negatively regulate cell replication are called "tumor suppressor" genes, and when mutated lose their ability to suppress cell growth. Due to the dominant nature of oncogenes, they are easier to identify and isolate. Unfortunately, tumor suppressor genes are more difficult to identify. Thus, at present the number of isolated oncogenes is greater than the number of tumor suppressor genes.

Our goal in this project is to utilize a new experimental approach for cloning tumor suppressor genes from mammary gland cells. To achieve this, we will employ the tools used by evolution. Our approach relies on the generation of diversity followed by selection. In addition, this approach parallels a strategy used by DNA tumor viruses to transform infected normal cells. Previous work has shown that these DNA viruses produce proteins that bind and inactivate the protein products of tumor suppressor genes. This stimulates cell growth to promote viral multiplication.

Following this viral-based strategy, we initially will select from a large collection of artificial bacterial viruses (known as phage libraries) that contain short, random stretches of amino acids (oligopeptides). We propose that these oligopeptides can specifically bind intracellular proteins of mouse mammary gland cells. Next, portions of the enriched, mammary gland-specific phage libraries will be introduced back into breast cells. The idea is to identify phage library-derived peptides that will inhibit undiscovered breast cancer tumor suppressors. We anticipate that the breast cells with inhibited tumor suppressors will have a selective growth advantage. Thus, our unique approach should circumvent the usual difficulties of identification and isolation of tumor suppressor genes. Finally, the isolated viral clones that inhibit tumor suppressors will be used to isolate their genes.

If this approach is successful in the identification and isolation of mouse mammary gland tumor suppressor genes, it will directly impact our understanding of human breast cancer. Any novel breast cancer tumor suppressor genes should contribute to the future development of diagnostic, prognostic and therapeutic approaches.

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Hormonal Control of HER2neu and BRCA1 in Breast Cancer

Wanda F. Reynolds, Ph.D.
Sidney Kimmel Cancer Center

The BRCA1 and the HER2neu genes are known to be important in the pathogenesis of breast cancer, but despite the efforts of many researchers, little is definitively known about how the control of these genes breaks down, or the mechanism by which estrogen might turn these genes on and off. The absence of BRCA1 protein in breast cancer cells is strongly linked to some familial breast cancers. An increased amount of HER2neu protein is associated with 30% of breast cancers, and correlates with poor prognosis and aggressive tumor growth. Considering the significance of these genes to breast cancer, there is a clear need to understand the mechanisms underlying this dysregulation, especially in regards to hormonal regulation of the amounts of HER2neu and BRCA1 protein.

There is evidence that estrogen influences the amounts of both HER2neu and BRCA1 protein in breast epithelial cells. Retinoic acid, a vitamin A derivative, and thyroid hormone are also implicated in the regulation of HER2neu. Our laboratory has obtained evidence that Alu elements associated with both HER2neu and BRCA1 genes may be responsible for this hormonal regulation. Alu elements are very abundant DNA elements, derived from a normal cellular gene termed 7SL. During the preceding 50 million years, the Alu elements have been replicated much like a virus and reinserted throughout the genome. During this time, for reasons as yet unknown, the Alu elements evolved to contain receptor binding sites. We found that the Alu elements associated with HER2neu and BRCA1 contain binding sites for receptors for retinoic acid, thyroid hormone, and estrogen receptor. We hypothesize that these hormones/vitamins associate with their receptors, which then bind to the DNA promoter sequences upstream of HER2neu and BRCA1 genes, thereby increasing or decreasing the amounts of HER2neu and BRCA1 that are made. We propose to determine whether these receptor binding sites are of key importance to the hormonally controlled dysregulation of HER2neu and BRCA1 in breast cancer.

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Fate Mapping of Progesterone Receptor Positive Breast Cells

G. Shyamala, Ph.D.
Ernest O. Lawrence Berkeley National Laboratory

Epidemiological studies have clearly established that, excluding the genetic background, reproductive history is an important and consistent "natural" risk factor associated with breast cancer. In accordance with this, experimental models have clearly established that the female sex steroids, estrogen and progesterone, are essential for both the development of the normal breast and the induction of breast cancer. It is also well established that cancers most often arise from the undifferentiated structures present most frequently in the breast of young females who have never been pregnant.

The breast is composed of many cell types including the epithelial cells, which are the targets for normal growth and are the ones most likely to give rise to cancers. In a normal breast, the epithelial cells grow in response to progesterone e.g. during the progesterone dominant phase of the menstrual cycle and pregnancy. More importantly, it is during pregnancy that the undifferentiated structures of the breast are converted to their differentiated counterparts. Using genetically engineered mice, our laboratory has direct proof that this conversion of undifferentiated structures to differentiated structures requires progesterone and progesterone receptors, the protein through which progesterone action is mediated. We have also found that epithelial cells that contain the progesterone receptor are a distinct subset. Thus, we believe that the loss of progesterone receptor positive cells, or a loss in the ability of progesterone receptor to convert undifferentiated cells to a differentiated state, increases the sub-population of cells at risk for breast cancer. Our long term objective, therefore, is to follow the fate of progesterone receptor positive cells as they undergo normal development during pregnancy and when they are exposed to cancer risk. For this, we need to create animal experimental models in which we can follow the fate of epithelial cells containing progesterone receptor and their negative counterparts on a cell by cell basis in a heritable manner. At present, such a system does not exist. Therefore, the goal of our project is to create genetically engineered mice that can be used to meet the long term objectives of this grant.

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Recombination and Mutation in Breast Cancer

David A. Zarling, Ph.D.
Pangene Corporation

Breast cancer is associated with numerous genetic alterations that can be detected directly in the chromosomal DNA by methods that visualize the attachment of probes labeled with fluorescent markers. This standard method of fluorescent in situ hybridization (FISH) is time consuming, insensitive and is not allele specific. Our lab has recently developed an enhanced FISH method for the detection of a tumor suppressor gene (p53) and an oncogene (ERBB2) in human breast cancer. Our method is rapid (30 min), sensitive, and appears to have a very low background. We plan to adapt our recombinase-enhanced hybridization method with novel ‘cyclizable’ DNA probes to develop an allele-specific DNA hybridization method for the specific and rapid detection of mutant breast cancer genes. To develop and apply this novel technology to breast cancer, we will employ a collaborative multidisciplinary research program combining the expertise of three laboratories: Pangene Corporation, the University of California at San Francisco, and Uppsala University in Sweden.

A second objective of this grant is to study the status of recombination (exchange of strands of homologous chromosomal DNA) genes and proteins in breast cancer cells. We have made preliminary observations that one of the key genes involved, RAD51, is abnormally regulated in breast cancer cells. We plan to expand on this observation and understand the molecular basis for the abnormal regulation of RAD51 and its potential role in the onset and progression of breast cancer. In related experiments, we will evaluate the possibility of using RAD51 as a marker for breast cancer diagnosis. Recently, the p53 tumor suppressor protein was reported to interact with the RAD51 protein and control its biochemical functions, including DNA strand exchange. In contrast, mutant p53 fails both to interact with RAD51 protein and to control DNA strand exchange. These related observations, together with the fact that breast cancer cells often have mutant p53 and abnormal rates of recombination, strongly suggests that abnormal regulation of recombination enzymes exist in breast cancer.

Thus, the goals of our research are to (i) provide a rapid method of evaluating breast cancer biopsy samples to detect genetic abnormalities, and (ii) explore novel mechanisms by which already known mutations are associated with DNA repair and recombination defects. These novel techniques offer the prospect of tailoring therapy options to the particular cellular and genetic defects of individual patients.

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Postdoctoral Fellowship Awards I

 

Molecular Analysis of BRCA1 Function

Mark S. Chapman, Ph.D.
Salk Institute for Biological Studies

A gene called BRCA1 normally acts to prevent breast and ovarian cancer. Individuals with mutations (genetic changes) in this gene have a very high risk of breast cancer, as well as ovarian cancer. The BRCA1 gene was isolated two years ago, which allows for study of the BRCA1 protein that the gene encodes. Currently, it is not known exactly how BRCA1 prevents cancer. It appears to act by controlling cell growth in breast tissue, and in the ovaries, since uncontrolled cell growth results in cancer. I have shown that the BRCA1 protein may be a transcription factor, which is a type of protein that regulates how much of certain other proteins are made. Since BRCA1 is such an important gene in breast cancer, it is important to know how it works. To show that BRCA1 works as a transcription factor, I need to show that this function is important for BRCA1 to control cell growth. I will do this by using cells grown in flasks and making changes to BRCA1 that affect its function as a transcription factor. I will then ask whether these changes also affect how BRCA1 controls cell growth. If BRCA1 is really a transcription factor, then changes that make it a worse transcription factor should also make it worse at controlling cell growth. Similarly, changes that make BRCA1 a better transcription factor should make it better at limiting cell growth. I will also try to identify which proteins might be regulated by BRCA1, since these genes may also be important for preventing breast cancer.

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Breast Carcinoma Associated MAR-Binding Proteins p90 and p30

Sanjeev Galande, Ph.D.
Ernest O. Lawrence Berkeley National Laboratory

The nucleus of a typical mammalian cell contains an enormous length of DNA, which is "packed" into a space that is about 10 microns in diameter. The nuclear matrix contributes to the structural and functional organization of DNA by providing the skeletal framework onto which the DNA is assembled into independent looped domains. Our research interest is focused on specific DNA sequences called MARs (matrix attachment regions), that presumably anchor DNA onto the nuclear matrix. Presence of cell type-specific proteins that bind MARs strongly suggests the role of MARs and MAR-binding proteins in cell development and differentiation. Recently, it has become appreciated that the structure and composition of nuclear matrix is altered in cancer cells, and this may affect the activity of genes that induce tumor formation.

In these studies, we will examine two new MAR-associated proteins from breast tumor cells. These proteins appear to be more abundant in breast tumor cells, such that virtually no MAR-binding can be detected in normal breast tissues or non-malignant breast lesions. These observations suggest that the two MAR-associated proteins of the cell nucleus may play an important role in the process of tumor formation. We propose to clone the cDNAs (genes) that encode for these two proteins using breast cancer cells. For studies on cellular distribution and quantitation, we will also prepare antibodies against the two purified MAR-associated proteins. Both the cloned genes and the antibodies will form essential tools for studying the function of these proteins at the molecular level. One hypothesis we wish to test is that inhibition of expression of these MAR-binding proteins or of their association with MARs will revert breast cancer cells to the normal phenotype. These studies will advance our knowledge on the mechanisms underlying breast tumor progression, and they will make possible the development of novel diagnostic markers for early detection of breast tumors.

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Characterization of Heregulin Targeted Genes

Fabiana Guerra-Vladusic, Ph.D.
Ernest O. Lawrence Berkeley National Laboratory

The growth of cancer cells depends in general on small proteins called growth factors, that bind to and turn on cellular growth factor receptors. One of these growth factor receptors, erbB-2, plays an important role in breast cancer and is present in higher amounts than normal in 30% of breast cancer patients. However, it is known that 70% of breast cancers with abnormally high amounts of erbB-2 are classified as non-invasive intraductal carcinomas, which are generally less aggressive breast cancers. This suggests that abnormally high amounts of erbB-2 on its own may not be sufficient to cause cells to form more aggressive tumors and to spread to other parts of the body. It is possible that additional growth factor receptors such as the recently cloned erbB-3 and/or erbB-4 receptors, may be required for the induction of invasive properties through receptor dimerization. The identification of HRG (a growth factor that binds and turns on the erbB receptors) has allowed us to characterize specific effects of the HRG/erbBs system, on cell growth, differentiation, and invasion. We have shown that HRG at low concentrations increases the invasive phenotype of breast cancer cells. Paradoxically, relatively high concentrations of HRG induces differentiation of erbB-2 overexpressing breast cancer cells.

We hypothesize that the distal effects induced by HRG (cell proliferation and/or differentiation) are probably due to the activation or expression of a number of different genes. As a result of our studies, we expect to gain a better knowledge of the genes that are triggered by HRG action. Once these genes have been identified, we can determine more effective methods for reversing or blocking the abnormal growth. Thus, we predict that our studies will lead to the development of specific therapies for breast cancer.

This model will allow us to identify positive and/or negative growth regulators involved in HRG induction of the distal biological effects. Therefore, the aims of our proposal are:

1. To determine if the nuclear localization of HRG is mediated by a mechanism dependent of the erbBs receptors.

2. To isolate genes that are differentially expressed as a result of HRG induction of cell growth and/or differentiation.

3. To determine the contribution of these genes as supressor- or promoter-like activities.

The results from these studies will provide a better understanding of HRG action and its distal biological function. If successful, these results will serve as a foundation for developing novel therapeutic approaches and prognostic data for breast cancer.

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Integrin Activation in the Metastasis of Breast Cancer

Emme Lin, Ph.D.
The Burnham Institute

In breast cancer, the most dangerous stage of the disease is when the tumor cells spread (metastasize) to other areas of the body. When this happens, the chance for survival is decreased because tumor invasion can be very widespread. Integrins are proteins on the cell surface that are involved in cellular adhesion functions necessary for attachment and movement. Cells have several integrins on their surface, and the presence and role of specific integins in tumor metastasis is not clear. The focus of our research is on a specific integrin receptor, called avb3. In previous work we have found that it is present at higher levels in breast cancer tumors that are metastatic. We showed direct evidence that avb3 is necessary for the invasion of breast cancer.

Our research has two objectives. First, we propose to strengthen the observation on the essential role of avb3 in metastasis. For these experiments we will use mutant forms of avb3 and examine the ability of cells to form tumors in mice. Secondly, we will test the hypothesis that the cell adhesion function of avb3 is regulated by an "On/Off switch" that operates within the cell. Our hypothesis is that when avb3 is "on," the cell will bind tightly to its surroundings such that the cell is immobilized. Conversely, when avb3 is "off," the tumor cell can "walk away" and spread to other areas of the body. Normal cells can regulate the "On/Off switch," so that the avb3 function becomes modulated. Thus, tumor cells gain the ability to invade because they can no longer control the "On/Off switch." Our approach will be to (i) develop antibodies to "freeze" the avb3 integrin receptor in a state to prevent metastasis and (ii) study a possible regulatory protein for the "On/Off switch." The regulatory protein is apparently absent in tumor cells, which allows them to spread. We will test this by introducing this regulatory protein into tumor cells to determine whether metastasis is affected.

These studies will possibly provide new breast cancer therapeutic targets (the "On/Off switch" and its regulators) for designing drugs to halt the spread of breast cancer.

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EMC-Regulated Transcription in Breast Cancer

Philippe Pujuguet, Ph.D.
Ernest O. Lawrence Berkeley National Laboratory

It is easily appreciated how the environment can effect the function and appearance of a living organism. An analogous process occurs at the level of an individual cell interacting with its local environment. Although the different cells in the body have the same genetic information in their DNA, they become specialized for various functions. A critical factor in maintaining a cell’s specialized structure and function is the immediate surrounding environment. As proof of this, cells upon removal from their natural habitat in a living organism lose their morphological and functional differences. Our laboratory studies the extracellular matrix (ECM), which is a fibrous network of proteins and glycosaminoglycans that forms the immediate environment of a cell. After properly ‘sensing’ its environment, a cell assumes the normal physiological state and mutually interacts with the ECM to create a state of ‘dynamic reciprocity’. The specific cell surface receptors involved in sensing this reciprocity are the ‘integrins’, which trigger the critical signaling events within the cell. The ‘processing’ of the ECM-integrin interactions regulates the three available options for a cell: (i) growth/division, (ii) changes in function (differentiation), or (iii) death (apoptosis).

We are pursuing the hypothesis that ‘incorrect sensing’ of this ECM signaling is a hallmark of cancer cells. Normal breast epithelial cells are in contact with a ‘basement membrane’ that consists of ECM molecules. We have found that this cell-ECM association regulates specific genes. An almost complete reversion of breast tumor cells to a normal breast cell phenotype has been obtained in a cell culture model, by blocking incorrect ECM signaling in the tumor cells. This fellowship is focused on the mechanism of ‘ECM responsiveness’ of normal mammary epithelial cells and ‘ECM-unresponsiveness’ of breast tumor cells. I will examine specific pathways in the cell that link the ECM-integrin interactions at the cell surface with the regulation of genes through so-called ‘transcription factors’. We have identified the ‘transcription factors’ that allow a normal mammary cell to function appropriately to produce differentiation (milk) proteins, and now are in a position to determine how this process is disrupted in tumor cells. Our eventual goal is to regain ‘ECM-responsiveness’ that could either reverse or inhibit the invasive/unregulated growth processes that characterize breast cancer cells by manipulating these ‘transcription factor’ pathways.

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Identification of Proteins Modulating Integrin Activation

David M. Rose, D.V.M., Ph.D.
The Scripps Research Institute

The major factor contributing to the morbidity and mortality of breast cancer is the metastasis and invasion of distant sites by breast carcinomas. The process of metastasis and invasion of tumor cells requires that these cells alter their ability to adhere to both surrounding cells and the extracellular matrix (ECM). Integrins are a family of cell surface proteins that anchor cells to specific component proteins of the ECM. The integrin receptors are known to be modulated quantitatively and qualitatively in cancer. We are interested in the qualitative modulation (i.e., affinity towards ECM component proteins outside the cell) of integrin function, which is thought to occur through the binding of other proteins inside the cell. We hypothesize that changes in integrin affinity towards the ECM can control the invasive and metastatic properties of breast cancer cells.

We propose to identify and characterize breast cancer proteins that are involved in integrin affinity regulation. Our laboratory has developed a novel approach, which utilizes Chinese hamster ovary (CHO) cells having modified integrin receptors. Introduction of isolated ‘interior portions’ of integrins into cells suppresses the activation status of these receptors. Our strategy is to introduce additional copies of regulatory elements into these cells to ‘rescue’ integrin activation, and determine their effect on cell adhesion function. Thus, we can indirectly address the protein structural and sequence elements necessary for integrin affinity regulation. Our goal is to define these elements in breast cancer cells. We have already found a possible integrin regulatory protein in CHO cells. It is possible that either (i) this protein is also found in breast cancer cells or (ii) our approach can identify additional unique regulatory proteins in breast cancer. The characterization of these proteins that regulate cell adhesion will aid in the understanding of metastasis and invasion of breast carcinomas. The proposed work relates directly to the pathogenesis of breast cancer and may provide new diagnostic and therapeutic targets to aid in the detection and treatment of metastatic breast cancer.

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Postdoctoral Fellowship Awards II

 

Disruption of Cyclin E/Cell Cycle in Breast Cancer Cells

Kevin Sato, Ph.D.
The Scripps Research Institute

The ability of a cell to divide is highly regulated, and this regulation becomes defective in cancer. The progression (temporal and physical) of a cell through cell division is termed the ‘cell cycle’. Numerous genes have been identified that control the cell cycle. A critical phase is the transition from either a stationary (G0) or a growing cell (G1) into the phase of active DNA synthesis (S) immediately prior to cell division. The cellular protein factors that regulate the forward progression of the cell cycle are called cyclins. Mutations in the genes encoding cyclins have been detected in clinical breast tumors. In normal cell growth and division, the temporal expression and destruction of specific cyclins coordinate cellular activities required for ordered cell cycle passage. Significantly, it is during the first phase of the cycle, G1, that the cell makes the commitment to either (i) exit the cycle to differentiate, or (ii) begin DNA replication (S). Our research focus is the G1-S transition, which appears to be regulated by a specific cyclin, cyclinE (cycE). Mutations in the cycE gene have been observed both in cultured human breast cancer cells and in clinical breast tumors. Also, comparing normal human breast cells and breast tumors showed that cycE protein either was either present at a high level or was abnormally stable in the tumors.

Our research will further define the role of cycE activity and the proteins it regulates in breast cancer. The most direct means of studying cycE is to deplete the cells of the protein and compare them with normal cells for cell cycle abnormalities. The key approach we will use is to block cycE protein production at the RNA level using two types of inhibitory molecules called either ‘antisense’ or ‘ribozymes’. These inhibitory molecules block the production of cycE protein, and we anticipate a possible reversion of the breast cancer phenotype to more normal cells. The cell systems used in this project include both normal and tumorigenic human breast epithelial cells.

An improved understanding of cell cycle regulatory genes is essential to explain how carcinogens and genetic mutations cause uncontrolled tumor cell growth. Ultimately, this will greatly aid in the design and development of measures to prevent the initiation of breast cancer and therapies to combat it once it has developed.

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Regulation of Wnt: Clues for Breast Cancer Pathogenesis

Heidi Theisen, Ph.D.
University of California, Irvine

Genes that direct normal development are also involved in controlling cell division. Abnormalities in the regulation of these genes have been linked to breast cancer development. For example, the Wnt gene is required for normal development, but alterations in the amount of Wnt protein it makes are associated with abnormal proliferation in human breast tissue. In order to perform its function, Wnt turns on a series of other proteins in the cell. The interruption of any stage of this process could have an effect on the growth of cells. Another protein that is associated with breast cancer development, TGFb, can both regulate and be regulated by Wnt. An understanding of how these genes function to regulate cell division may elucidate how tumor cells proliferate and invade host tissues.

In this project, I will analyze what occurs within cells in response to Wnt and TGFb. I will look at the effect on cellular proliferation when each protein of the series is blocked from being turned on. I will also examine how proteins turned on by Wnt block TGFb function, and how proteins turned on by TGFb block Wnt function. Understanding the interaction of Wnt and TGFb could conceivably permit the development of strategies for designing improved methods for breast cancer intervention and treatment.

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Control of DNA Replication in Breast Cancer

Dieter Wolf, M.D.
Stanford University

The vast majority of human breast cancers have an increased, abnormal DNA content (ploidy) per cell. Increased ploidy caused by gene amplification has been shown to play an important part in the pathogenesis and prognosis of various solid tumors. The exceptionally high frequency of gene amplification in human breast cancers suggests that events that increase the DNA content play an important role in the genesis and progression of this disease. Our working hypothesis is that alterations in factors controlling DNA synthesis are important for the development of human breast cancers.

In previous work we have identified a human gene called ORC1, which has the capacity to cause an increase in ploidy in experiments we conducted using yeast cells. ORCI and its relative Cdc6 are both part of a large protein complex that regulates the initiation of DNA synthesis. The ordered assembly and disassembly of the ORC/Cdc6 complex appears to be necessary to ensure that each segment of the genome (all the chromosomal DNA of a cell) is duplicated only once during each cell division cycle. Defects in this process may lead to increased amounts of cellular DNA, or ploidy.

The work here focuses on the specific roles of ORCl and Cdc6 in the ploidy increases in breast cancer. We will perform this work on breast cancer and other mammalian cells in culture, and using protein extracts from amphibian eggs. We will determine both the potential and the structural requirements of these proteins to induce DNA ploidy increases in mammalian cells. Our strategies will include: (i) a detailed quantitative and qualitative analysis of ORC1 and Cdc6 in breast cancer cells to uncover potential increases in protein abundance or aberrant cell cycle timing of their interaction with nuclear structures, (ii) overproduction of ORC1 and Cdc6 in mammalian cells and examination for increased DNA content or cellular transformation, and (iii) a biochemical characterization of Cdc6 in the Xenopus (amphibian) in vitro DNA replication system, in order to study the role of purine nucleotide binding and hydrolysis in its replication function.

Despite numerous recent advances in the genetic identification of breast cancer susceptibility genes, there is a fundamental gap in our knowledge regarding global chromosomal changes. The novel features of our approach promises a potential breakthrough that could eventually allow for either the screening for or rational design of inhibitors that may be valuable in breast cancer therapy.

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TRAF-Regulated Signal Transduction in Breast Cancer

Juan Zapata, Ph.D.
The Burnham Institute

It is believed that potentially cancerous cells arise in our body nearly every day. Luckily, the cells of our immune system can usually eradicate these abnormal cells before they form lethal tumors. The goal of this project is to provide new insights into some of the mechanisms used by immune cells to attack and kill cancerous cells. One route used by immune cells to attack tumors is to produce proteins that bind to receptors on the target cancer cell. These receptors, in turn, deliver signals inside the tumor cells that activate a latent program for cell suicide. Understanding the molecular pathways that these suicide receptors initiate in the cancer cell has the potential to provide novel insights that might be exploited in clinical treatment strategies.

Some of the most important suicide receptors of cancer cells are members of the Tumor Necrosis Factor (TNF) Receptor family. Little is known, however, about how these receptors deliver signals into tumors to kill them. Recently, several laboratories have discovered a group of closely related proteins that interacts with TNF-family receptors, called TNF-Receptor Associated Factors (TRAFs). In preliminary studies one of these factors, TRAF-4, was found to be present at lower levels in breast cancer tumor samples compared to normal breast samples. This strongly supports our initial hypothesis that cancer cells are defective in their ability to respond to immune attack. Thus, the purpose of this research is to understand the mechanisms by which these TRAF-proteins transfer signals from the suicide receptors in breast cancer cells. We plan to use a variety of approaches, such as generating specific antibodies to TRAF proteins and performing analysis on their distribution and amounts in breast cancer specimens. We also plan to use molecular biology approaches to understand how the TRAF proteins both affect gene regulation and trigger cell suicide. The results of these investigations may provide insights leading to novel therapeutic approaches for breast cancer treatment and prevention. Our work could provide information to allow immune cells to better cope with breast cancer using the normal defense methods in the body.

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