Pathogenesis: Understanding the Disease
Researchers in breast cancer tumor biology are seeking answers to many key questions. How are breast cancer cells different from normal breast cells? How do breast cancers escape the limits of growth placed on normal cells? What are the critical underlying genetic characteristics for the major types of breast cancer? Why do breast cancer cells fail to respond to therapies and the body's own immune system? How do breast cancers gain a blood supply and spread in the body? These questions are being addressed at the cellular, molecular and genetic levels using BCRP funding. The research grants summarized below generally employ the modern tools of molecular biology to understand the unique genes and protein interactions that allow breast cancers to grow, progress, and spread in the body.
We divide the pathogenesis priority area into five broad sub-topics:
- Outbreak--How Cancer Spreads: Angiogenesis, Invasion, and Metastasis
- Too Much Cell Growth: Defective Messages and Internal Signaling
- Mistakes on the Master Blueprint: Molecular Genetics and Gene Regulation
- Searching the Unknown: Novel Breast Cancer Genes
- Unraveling the Path to Breast Cancer: Tumor Progression
Research Conclusions Research in Progress Research Initiated in 2000 |
Research Conclusions
Outbreak--How Cancer Spreads: Angiogenesis, Invasion, and Metastasis
Balance of Growth Factors in Breast Cancer Growth and Metastasis Daisy De Leon, Ph.D., of Loma Linda University examined the role of two proteins, IGF-II and Cathepsin D, in breast cancer spreading to other parts of the body. She found that when breast tumor cells are exposed to the precursor form of IGF-II, they move and divide more. IGF-II causes both hormone-dependent and hormone-independent breast cancer cells to release Cathepsin D. Dr. De Leon's theory is that the release of Cathepsin D correlates with the breast cancer moving to other body parts. Dr. De Leon found that of the 5 to 6 different forms of Cathepsin D, only one was elevated in breast cancer tissues.
This investigation showed that Cathepsin D and IGF-II are good candidates to serve as markers for tumors that are likely to spread to other body parts. Results from this funding were reported in three publications, including Hormone Metabolism Research 31:142-7 (1999).
Novel Breast Cancer Epithelial Cell Metalloproteinase
Pierre-Yves Desprez, Ph.D., from the California Pacific Medical Center, San Francisco planned to clone and study an invasion protease, metalloproteinase. An invasion protease is a protein that digests a cell's immediate environment and allows the cell to move. Metalloproteinase is secreted from breast cells under the control of Id-1, a type of protein called a transcription factor. Using a polymerase chain reaction (PCR) cloning method, the research team identified an interesting zinc finger protein, a type of protein that regulates and binds to DNA. Although it was not a protease, this new protein did exist in gene/protein databases. When the novel zinc finger protein was re-introduced into breast cells, it induced processes associated with milk secretion, but not through any role as a transcription factor. Dr. Desprez is continuing this research to find the relationship of the novel protein with Id-1 and its function in breast cells.
Abnormal Regulation in Breast Cancer Development/Metastasis
Ulla Knaus, Ph.D., from The Scripps Research Institute, La Jolla investigated a signaling protein, PAK, that appears to be a key player in relaying growth messages inside breast cancer cells. Dr. Knaus found that PAK was associated with two other proteins, Rac and Cdc42. These three proteins are collectively involved with critical functions of cell movement and the cancer's spread in the body. Results from this research were published in the Journal of Biological Chemistry 273:8137 (1998) and the Proceedings of the National Academy of Sciences, USA 97:185 (2000). Dr. Knaus received additional funding from the BCRP in 2000 to continue this research.
Role of Gamma-catenin in a Breast Cancer Mouse Model
Normal breast epithelial cells have a recognition system that maintains their organization. This recognition system works, in part, through cell surface proteins called cadherins, which relay messages though "switchboard" proteins, called catenins. John Reed, M.D., Ph.D., from The Burnham Institute, La Jolla highlighted the mechanism by which two signaling proteins, called APC (a tumor suppressing protein first found in colon cancers) and Siah (the human counterpart of a family of Drosophiila eye development genes) cooperate to degrade catenins and limit cell growth. This research indicates that colon and breast cancers have similarities in this type of cell regulation. A portion of this work was published in The Journal of Biological Chemistry 275:15578-15585 (2000). Dr. Reed's team plans to pursue these findings, using animals with cloned genes transferred into their DNA that contain defects in key elements of the cell death (apoptosis) process.
Too Much Cell Growth: Defective Messages and Internal Signaling
Degradation of Growth Factor Receptors and Breast Cancer
Epidermal growth factor receptor (EGFR) and Her-2/neu oncogene receptor are two proteins that are often present in abundant amounts in breast tumor cells. Cells constantly recycle their receptors, and in normal cells the balance of receptor production and degradation is tightly regulated. Although intensive research has been undertaken to understand how receptors are overproduced in cancers, few studies have been done on how receptors are degraded. Gordon Gill, M.D., of the University of California, San Diego investigated the degradation side of the equation. The cell system that directs degradation of EGFR uses a protein, SNX1, which is a member of the sorting nexin family of proteins. SNX1 recognizes a part of EGFR molecule. Dr. Gill found a second, previously undiscovered, related protein called SNX2 and, since then, 16 additional members of this protein family have been discovered. Dr. Gill found that SNX1 and SNX2 work together to bring EGFR into the part of the cell where it can be degraded by the lysosomes. Dr. Gill examined the structure of the sorting nexins and found that one part (the COOH terminus) is responsible for directing EGFR into the lysosomes and another (the PX domain) is responsible for interactions between SNX proteins. Continuation of the study may open up new approaches for stabilizing the receptor levels in breast cancer. This could make the cancer cells behave more like normal cells. The results were reviewed in Current Opinions in Cell Biology 11:483-8 (1999).
Role of Rb Protein and Cell Cycle Defects in Breast Cancer
The retinoblastoma gene (Rb) has been investigated in many cancer types. It serves as a regulatory switch, turning off and on numerous key genes that keep cells from multiplying. Mutations in Rb or defects in other proteins that regulate Rb release cells from normal controls and have been found in breast and other cancers. Kathryn Ely, Ph.D., at The Burnham Institute, La Jolla studied a protein that binds with Rb called RIZ. She focussed on a part of RIZ called the PR domain. The Ely laboratory conducted crystallography, nuclear magnetic resonance, and mutational analysis of the PR domain. In other published work, it has been reported that breast cancer cells produce a modified RIZ protein that lacks the PR domain. Thus, understanding how PR works and its structure could be important to restoring normal cell functions in breast cancer.
The Role of a Novel Estrogen Receptor in Breast Cancer
Ruth Lupu, Ph.D., at the Lawrence Berkeley National Laboratory, Berkeley explored the function, in normal and tumor breast cells, of a newly discovered protein, an estrogen receptor called estrogen receptor beta (ER-b). She found that ER-b was detectable in 20% of 50 breast tissues examined. It inhibits cell growth when introduced into cells that don't have estrogen receptors. Dr. Lupu also identified a variant of ER-b in these studies called ER-b5 that appears to inhibit ER-b's action. Additional details of the study can be found in Oncology Reports 7: 157-167 (2000).
Hormonal Control of HER2neu and BRCA1 in Breast Cancer
Unraveling how two genes involved in breast cancer, Her-2/neu and BRCA1, are turned on and off would provide critical clues to how the disease develops. Wanda Reynolds, Ph.D., at the Sidney Kimmel Cancer Center, San Diego investigated the possibility that the Her-2/neu and BRCA1 genetic sequences have parts called Alu elements (AluHRE) which bind to hormones. If this is so, steroid hormones would turn the genes on and off. Dr. Reynolds found that the hormone estrogen bound to the AluHRE and increased the levels of both genes, but retinoic acid (Vitamin A) and thyroid hormone decreased the levels. The results support the theory that AluHRE plays a role in these genes getting turned on and off. One step in cells turning into cancer is breakage in their DNA. Dr. Reynolds investigated whether AluHRE were likely sites of DNA breakage, but found that this was not the case.
A Novel Drug Induces Apoptosis in Breast Cancer Cells
When chemotherapy fails for the treatment of advanced breast cancer, it is in part because the tumor cells have mechanisms to evade programmed cell death (apoptosis). Glenn Rosen, M.D., at Stanford University, Palo Alto found that a compound called Triptolide, derived from a Chinese herb, greatly enhances the effect of chemotherapuetic drugs. Triptolide works by suppressing the action of a protein from white blood cells, tumor necrosis factor (TNF). TNF turns on a series of protein interactions that inhibit cell death. In addition, Triptolide sends confusing messages for cell division; the cell responds by initiating cell death. Dr. Rosen reported this research in two publications in the Journal of Biological Chemistry 274:12451-13455 (1999) and 276: 2221-2227(2001). He is continuing these studies to determine the molecular site of Triptolide's action in breast cancer cells, and has applied for a patent for this approach in chemotherapy.
Fate Mapping of Progesterone Receptor Positive Breast Cells
The hormone progesterone and its receptor (the protein that allows cells to take it up) are intricately involved in guiding the development of the breast. However, the mechanism is unclear. G. Shyamala, Ph.D., at the Lawrence Berkeley National Laboratory, Berkeley attempted to determine how cells that have progesterone receptors become distributed during breast development. She made genetically engineered mice that carried a 'tagged' progesterone receptor in the mammary gland. Due to unexpected difficulties, Dr. Shyamala was unable to follow the distribution of the progesterone receptor-tagged cells. However, she did find that the mammary glands in these mice grew much more slowly than their normal counterparts. This indicates that a part of the progesterone receptor can impede normal breast development.
Regulation of Wnt: Clues for Breast Cancer Pathogenesis
Heidi Theisen, Ph.D., of the University of California, Irvine resigned for medical reasons before this study was completed . The goal of the project was to determine the series of genes that get turned on and off in cell growth, beginning with a protein made by a gene called Wnt. Dr. Theisen was examining the interaction between Wnt and a protein called TGF-b during cell division in the simpler developmental system of flies. She was attempting to pinpoint genes that behave similarly in human breast cancer development. She was able to develop the DNA she needed to insert into the flies before she had to resign.
The Regulation of p53 Activity in Breast Cancer
Yang Xu, Ph.D., from the University of California, San Diego resigned before this study was completed. Proteins in cells can be activated or de-activated when a molecule of phosphorus is added to them (phosphorylation). Dr. Xu planned to study the parts of a protein, tumor suppressor p53, where the phosphorus molecules can be added. Processes that lead to cell death could be influenced by changes in p53 phosphorylation specific to breast cancer. Even though most breast cancers appear to have normal p53, their p53 could have defects that keep them from attaching to the phosphorus molecule that would lead to cell death and other responses. Research begun with BCRP funding was published in the EMBO Journal 15:19 (2000).
Mistakes on the Master Blueprint: Molecular Genetics and Gene Regulation
Molecular Analysis of BRCA1
Mark Chapman, Ph.D., at the Salk Institute for Biological Studies, La Jolla investigated the ways in which the BRCA1 gene is involved in regulating other genes in the cell. When a woman has mutations in the BRCA1 gene, she runs a higher risk of breast cancer. Dr. Chapman studied the normal BRCA1 genes, which do not have the mutations that make breast cancer more likely. His hypothesis was that normal BRCA1 genes inhibit cell growth by turning on the p53 pathway, a complex process that eventually causes cell death. However, he found that BRCA1 is triggering additional processes that are more important than any action it causes by turning on p53. Dr. Chapman found a previously unidentified protein that interacts with both BRCA1 and BRCA2 genes and appears to amplify the effects of the genes.
Biochemical and Functional Characterization of BRCA1
The key hereditary breast cancer gene 1 (BRCA1) was cloned in 1994, but researchers haven't yet learned what the gene does or how it is regulated. Heinz Ruffner, Ph.D., also from the Salk Institute for Biological Studies, La Jolla published a key study (Molecular and Cellular Biology, 19:4843, 1999) that demonstrated an interaction of a protein that regulates the normal process of cell growth and division, CDK2, with the BRCA1 protein. Apparently, CDK2 adds a molecule of phosphorus to the BRCA1 protein at a location on the protein structure known as serine residue 1497. Adding a molecule of phosphorus generally turns off or on a protein's function. Dr. Ruffner is continuing to investigate the function of CDK2's modification of BRCA1.
Siah-family Genes: Effectors of p53 in Breast Cancer
Simple organisms, such as fruit flies, yeast, and nematodes, have yielded clues to important cell regulatory genes that might be relevant to cancer biology. Drosophila (fruit fly) and Caenorhabditis (nematode worm) are animals that have strong regulatory mechanisms to control cell division. These mechanisms could be used to halt breast cancer growth. Shu-ichi Matsuzawa, Ph.D., at The Burnham Institute, La Jolla explored the role of Siah genes, which are human counterparts of a family of Drosophila eye development genes. Dr. Matsuzawa found that Siah and proteins that interact with Siah work through a group of proteins that control the stability of an important protein, called b-catenin. b-catenin maintains the integrity of the normal epithelial cell layer. Most breast cancer develops in the breast's epithelial cells. Degradation of b-catenin allows tumor cells to escape control of the structure around them and to grow as cell masses. This process is better understood for colorectal cancer, but is also important for most breast cancers. Part of this research was published in the EMBO Journal 17:2736-2747 (1998).
Genes Involved in Multistep Mammary Tumorigenesis
Gregory Shackleford, Ph.D., of the Children's Hospital, Los Angeles took steps toward creating a mouse model that would identify genes involved in breast cancer development and serve as a tool to dissect the ways in which the genes interact. Dr. Shackleford genetically engineered mice to carry an inhibitor of a protein produced by breast cells, a growth factor called FGF (fibroblast growth factor). He mated these animals with a different line of genetically engineered mice carrying a gene, Wnt10b. Genetic pathways are combinations of genes that initiate some process in cells when they are turned on or off. Through future experiments, the team will determine whether the genetic pathways associated with FGF and Wnt10b work together to affect the growth of breast tumors and ultimately identify other genes involved in this process. Dr. Shackleford was co-author in a study reporting key findings from this funding, Oncogene 17:2711-2717 (1998).
Alteration of Developmental Genes in Breast Cancer
Fumiichiro Yamamoto, Ph.D., of The Burnham Institute, La Jolla investigated ways that genes can be turned on or off by a process called methylation. He also investigated developmental genes, called homeotic genes, that perform functions that are remarkably similar to those involved in tumor development. The goal was to determine whether developmental genes that are regulated by methylation play a role in tumor development. Dr. Yamamoto found that methylation is different in some homeotic genes, such as Hox B13 and IPF-I, in most breast cancer cases, when compared to normal tissue. However, these differences did not translate into mutations on the genes or changes in the levels of proteins the genes produced. Methylation inhibitors were able to decrease Hox B13 levels, and had varying effects on the levels of other homeotic genes. These results indicate that DNA methylation inhibitors may be poor candidates for use in breast cancer therapy.
Searching the Unknown: Novel Breast Cancer Genes
Identification of New Candidate Breast Cancer Genes
Breast cancer cells undergo dramatic deletions, duplications, and rearrangements of their chromosomal DNA, and these account for some of the increases and decreases in the amounts of gene-produced proteins measured in tissue samples from tumors. Donna Albertson, Ph.D., first at Lawrence Berkeley National Laboratory, then at University of California, San Francisco, used a microscopic, direct visualization technique called fluorescence in situ hybridization (FISH) to examine breast cancers and identify novel genes and chromosomal regions. This search led to the discovery of a gene called ZNF217 that appears to be a key player in the immortilization of breast cancer cells, the process that allows tumor cells to keep dividing after they have completed the normal number of divisions for breast cells (about 100). A gene physically associated with ZNF217, called CYP24, also appears to be important in breast cancer. Three publications were supported by this funding with the most recent in Nature Genetics 25:144-146 (2000). Dr. Albertson and colleagues are continuing to analyze the gene sequence of breast cancer using another technique, gene expression microarrays, which yields more information in a smaller amount of time.
Breast Carcinoma Associated MAR-binding Proteins p90 and p70
Sanjeev Galande, Ph.D., from the Lawrence Berkeley National Laboratory, Berkeley investigated how the attachment of chromosomal DNA to proteins in the nucleus of breast cancer cells differs from normal cells. Differences in the attachment of DNA on the chromosome could underlie larger differences in gene structure seen in breast cancer. Working with his mentor, Dr. Terumi Kohwi-Shigematsu, Dr. Galande unexpectedly identified a protein complex, previously associated with DNA repair, that seems to play a role in making the DNA in cancer cells different from that of normal cells. This complex is composed of two proteins, called PARP and DNA-dependent-protein kinase (DNA-PK), and is seen in increased amounts in breast cancer cells. Results from this research were published in the Journal of Biological Chemistry 274:20251 (1999) and Critical Reviews of Eukaryotic Gene Expression 10:63 (2000). Dr. Kohwi-Shigematsu is receiving further funding from BCRP to pursue this work.
Unraveling the Path to Breast Cancer: Tumor Progression
Does Cell Aging Cause Breast Cancer?
Breast epithelial cells are the cells where most breast cancer begins. They co-exist in the mammary gland with other cells called fibroblasts, which form the connective tissue framework of the gland. As the body ages, the supporting fibroblasts become less functional, senescent (elderly) cells. Judith Campisi, Ph.D., from the Lawrence Berkeley National Laboratory, Berkeley showed that epithelial cells engineered to represent the early stages of breast cancer were stimulated by senescent fibroblasts, but not by normal fibroblasts. This shows that younger women have breast tissue barriers to prevent epithelial cells from developing into cancer. In contrast, older women have less functional control systems, which allows early cancers to emerge, divide, and go on to develop into more aggressive cancers. Dr. Campisi is continuing this work to identify the specific changes in aging fibroblasts that allow breast cancer, and to find the breast epithelial genes that normal fibroblasts most highly regulate.
Progesterone Receptor and Remodeling of Basement Membrane
The maintenance of mammary gland structure is important for the normal functioning of the epithelial cells (the cells both responsible for milk production and the origin of more than 95% of breast tumors). In the breast, epithelial cells lie on a complex structure called the basement membrane, which is composed partly of collagen. Factors that affect the integrity of the basement membrane can have a significant effect on the development of breast tumors. G. Shyamala, Ph.D., at the Lawrence Berkeley National Laboratory, Berkeley created genetically-engineered mice to test the hypothesis that the steroid hormone progesterone is involved in the integrity of the basement membrane. She found that mice with excess progesterone receptor-A lacked appropriate amounts of basement membrane components. This caused the epithelial cells to form abnormal structures. This study emphasizes the necessity for considering the effects of progesterone when devising hormonal therapies.
Proteolysis of Cyclin E in Normal and Malignant Breast Cells
Heimo Strohmaier, Ph.D., from The Scripps Research Institute, La Jolla studied the underlying molecular mechanism that allows breast cancer cells to have elevated amounts of a protein that promotes cell division, cyclin E. Using yeast cells as a model system, the team identified a protein called Cdc4, which appears to be a key factor that regulates the cell quantities of cyclin E. Next, they plan to identify the proteins and groups of interacting proteins that control cyclin E production in human breast cancer cells.
Research in Progress
Outbreak--How Cancer Spreads: Angiogenesis, Invasion, and Metastasis
Breast Cancer Cell Binding to the Endothelium. Brunhilde Felding-Habermann, Ph.D., from The Scripps Research Institute, La Jolla is finding that in the final stage of spreading to another part body, breast cancer cells appear to interact with cells in the blood called the platelets. Integrin receptors are substances found in both normal breast cells and cancer cells; in normal cells integrin receptors help keep the cell in place, but they play a role in the spread of breast cancer cells. Dr. Felding-Habermann found that the integrin receptor on breast cancer cells, called avb3, can exist in both an activated and de-actived state. Only the activated form of avb3 will support the interaction of breast cancer cells with the endothelial cells that line blood vessels, which is one step in cancer's spread. If ways of blocking this process were discovered, then breast cancer would be less capable of spreading.
Spatial Control of Matrix Proteolysis in Breast Cancer. Alex Strongin, Ph.D., at The Burnham Institute, La Jolla found that the integrin receptor avb3 appears to bind directly with a protein, the matrix metalloproteinase MMP-2, which is found in the structural framework that surrounds breast cells. This binding is one of the interactions that makes it possible for tumor cells to move from the breast into the blood and to other parts of the body. These results were published in the International Journal of Cancer 86:15-23 (2000).
How Does Endostatin Inhibit Breast Cancer Angiogenesis? Endostatin is an anti-angiogenic protein (a protein that inhibits the formation of blood vessels). A large amount of research is being done on it, because it eliminates cancer in mice without side effects or creating tumors resistant to it. However, researchers don't understand very clearly how it works. Kristiina Vuori, M.D., Ph.D., at The Burnham Institute, La Jolla reports that endostatin binds with two integrin adhesion receptors found on the surface of cells that line blood vessels. It appears that the endostatin-integrin binding is involved in some as-yet-undiscovered function of integrin receptors. The results of these studies are in press in the Proceedings National Academy Sciences, USA.
Too Much Cell Growth: Defective Messages and Internal Signaling
Role of the EphB4 Receptor Tyrosine Kinase in Breast Cancer. Elena Pasquale, Ph.D., at The Burnham Institute, La Jolla is investigating whether a protein called EphB4 makes tumors more aggressive. EphB4 regulates the normal developmental processes of cell adhesion and movement in human and animal embryos. It is also present in human breast cancer cell lines and in aggressive mouse tumors, as well as mouse tumors that are spreading to other parts of the body. Dr. Pasquale has made cells with decreased EphB4 activity. Because these cells are engineered to appear green, their location and movement can be directly visualized. Dr. Pasquale has also made the unexpected observation that increased EphB4 can stop cell growth.
GATA-3 Expression in Hormone Responsive Breast Cancer. The protein GATA-3 is elevated in tumors that grow faster when the hormones estrogen and progesterone are present. The chemotherapy drug tamoxifen works by blocking estrogen; apparently, GATA-3 is involved in tumors developing resistance to tamoxifen therapy. Ronald Weigel, M.D., Ph.D., at Stanford University, Palo Alto is investigating whether GATA-3 binds with the estrogen receptor (a protein that allows a cell to take up estrogen), if the binding decreases cell levels of GATA-3, and if lower levels of GATA-3 allow the cell to evade the effect of tamoxifen. He found that GATA-3 and the estrogen receptor do not bind directly, but they may interact through an intermediary protein. Loss of GATA-3 function does not appear to explain tamoxifen resistance, but Dr. Weigel is pursuing the idea that there may still be another breakdown in the GATA-3 mechanism of action.
Searching the Unknown: Novel Breast Cancer Genes
Role of DNA Damage Response Gene in Breast Cancer. Eric Brown, Ph.D., at the California Institute of Technology, Pasadena is studying a gene, called ATR, that may regulate how efficiently cells can repair DNA damage. Cells with DNA damage tend to turn into cancer. If ATR regulates cell repair of DNA damage, the gene could be critical for restraining the growth of cancer and pre-cancer cells.
Unraveling the Path to Breast Cancer: Tumor Progression
TGF-b Receptor Signaling and Breast Cancer. Signaling proteins turn on changes inside the cell after a receptor protein has bound to a substance from outside the cell. Kunxin Luo, Ph.D., at the Lawrence Berkeley National Laboratory, Berkeley discovered two new signaling proteins called SnoN and Ski. These proteins are able to block the cell growth inhibition caused by a protein called TGF-b. Dr. Luo is continuing to investigate how these proteins exert their control over TGF-b and their influence on normal and tumor breast cell development. v Role of a DNA Damage Response Gene in Breast Cancer. One step normal cells go through on the way to becoming cancer cells is immortalization, where they escape from the fixed number of divisions that limit the life of most cells. Martha Stampfer, Ph.D., at the Lawrence Berkeley National Laboratory, Berkeley originally set out to identify new genes that were activated or deactivated during the process of cell immortalization. However, during the course of the investigation, Dr. Stampfer noticed that the activation of a cancer gene called raf1 stops growth in mortal cells, but it pushes immortal cells further on the path toward becoming cancer. Her team is continuing to investigate why raf1 has these differing effects.
Research Initiated in 2000
Outbreak--How Cancer Spreads: Angiogenesis, Invasion, and Metastasis
Analysis of Genes Predictive of Breast Cancer Metastasis. Jeffrey Gregg, M.D., from the University of California, Davis is following up on an observation about experimental mouse tumors with an enzyme called phosphoinositol kinase 3 (PI-3 kinase). When PI-3-kinase is turned on, the tumors will spread more often to the lung than to other parts of the body. PI-3 kinase works by activating a series of other enzymes and proteins. Dr. Gregg is using several screening techniques to determine which of these other proteins are directly involved in the spread of cancer.
Profiling Serine Protease Activities in Breast Cancer and Identifying Breast Cancer Targets for Protease Inhibitors. Proteases are proteins that digest other proteins and allow cells to move. Benjamin Cravatt, Ph.D., and Yongsheng Liu, Ph.D., at The Scripps Research Institute, La Jolla are identifying and studying proteases specific to breast cancer. They are using a novel chemical strategy developed in Dr. Cravatt's laboratory, termed Activity-Based Protein Profiling (ABPP). This technique allows them to monitor not just the amount, but the activity of many proteases simultaneously in samples of whole cells, the experimental media surrounding the cells, and tissues. Dr. Cravatt and Dr. Liu will also try to understand why lower levels of estrogen receptor proteins make cancer cells less likely to spread.
Role of Matrix Metalloproteinases in Breast Tumor Initiation and Aggressiveness. Jimmie Fata, Ph.D., at the Lawrence Berkeley National Laboratory, Berkeley will also study how the type of protein called proteases plows a path for cancer cells to migrate. Dr. Fata will investigate a process called epithelio-mesenchymal transdifferentiation (EMT), which is associated with aggressive breast cancer. In this process, epithelial cells (the cell type responsible for more than 90% of breast cancers) become more like another type of cells called fibroblasts. In contrast to epithelial cells, fibroblasts are very mobile and less tightly associated with other cells. Thus, as epithelial cells become more like fibroblasts, they gain the ability to move and spread.
TGF-b3 and small GTPases in Invasive Breast Cancer. Vesa Kaartinen, Ph.D., of the Children's Hospital, Los Angeles is also investigating the EMT process where breast epithelial cells become more like fibroblasts, and become more able to move. Dr. Kaartinen is investigating the role in the EMT process of three types of protein: transforming growth factors (TGF-b3), adhesion molecules (integrins), and signaling molecules (GTPases).
Analysis of Angiogenic Pathways in Metastatic Breast Cancer. Elizabeth Hindmarsh, Ph.D., at The Burnham Institute, La Jolla plans to use gene chip microarrays, a technique that allows her to measure proteins produced by 10,000 genes at a time. She'll compare the proteins produced by genes--from normal tissues, breast tumors and breast cancer that has spread to various organs--to determine which genes are involved with the growth of blood vessels.
The Role of IL-8 and Its Receptors in Angiogenesis. Interleukin-8 (IL-8) is a substance produced in the human body known for its role in inflammatory diseases, where it attracts white blood cells into an area of tissue injury. Interestingly, IL-8 is produced by breast cancer cells. Ingrid Schraufstatter, M.D., from the La Jolla Institute for Experimental Medicine, will investigate whether IL-8 stimulates cells that line nearby blood vessels to promote the growth of blood vessels that nourish tumors.
Cell Adhesion and Drug Resistance in Breast Cancer. Breast cancer cells can sometimes avoid being killed by chemotherapy, but researchers don't know how. Kristiina Vuori, M.D., Ph.D., at The Burnham Institute, La Jolla will attempt to address this issue. She will examine whether the local micro-environment of breast cancer cells could provide a protein that either allows cancer cells to survive chemotherapy, or blocks the cell death process after chemotherapy.
Too Much Cell Growth: Defective Messages and Internal Signaling
The Control of Breast Cancer Cell Death. Daria Mochly-Rosen, Ph.D., from Stanford University, Palo Alto will study the protein kinase C (PKC) family of enzymes. She will identify the member or members of the PKC family that allow breast cancer cells to live and form tumors. Then, her lab will test compounds to inhibit these specific PKC enzymes, looking for potential new breast cancer drugs.
A Novel Signal Transduction Pathway in Breast Cancer. Previous research has identified a unique response mechanism breast cancer cells use to evade the body's immune system. A series of interactions between proteins and genes, called NF-kB appears to be involved. Yixue Cao, M.D., Ph.D., at the University of California, San Diego will use yeast as a model system to determine how NF-kB can become dysfunctional in breast cancer.
Anti-E-Cadherin Apoptosis of Inflammatory Breast Carcinoma. Mary Alpaugh, Ph.D., at the University of California, Los Angeles- School of Medicine will use a unique mouse model to study inflammatory breast cancer, an unusual type of the disease that invades many nearby blood and lymphatic vessels.
Novel Mechanisms of ErbB-2-Mediated Breast Cancer Metastasis. Her-2/neu is a gene involved in cell growth in some types of breast cancer. Richard Klemke, Ph.D., at The Scripps Research Institute, La Jolla will investigate how specific segments of the protein produced by the Her-2/neu gene activate interactions between other proteins inside breast cancer cells. He will also see if the interactions are the same in breast cancer cells that migrate to other body parts.
The Role of the BMK1-MEKK3 Pathway in Breast Cancer. Ta-Hsiang Chao, Ph.D., also from The Scripps Research Institute, La Jolla is investigating cell growth related to the Her-2/neu cancer gene. Dr. Chao will use a yeast system to identify and isolate a key protein that turns on a series of interactions between genes and proteins that makes cells grow. The goals are to design compounds that inhibit breast cancer cells, but not normal cells, and to better understand the molecular nature of these gene-protein interactions that may be involved with breast cancer spreading.
Studies on the Role of the ER-b in Breast Cancer. ER-a and ER-b are two estrogen receptors, proteins that allow cells to take up the hormone estrogen, triggering a process involved in cell growth. Eli Gilad, Ph.D., from the Lawrence Berkeley National Laboratory, Berkeley will explore situations where both ER-a and ER-b are present in the same cell, and see how they stimulate cell growth responses.
Cell Growth Control of Breast Epithelial Cells. Ulla Knaus, Ph.D., at The Scripps Research Institute, La Jolla will investigate the observation that a protein that is consistently active in human breast cancer cells, called Rac3, is required for tumor cells to grow and spread, while a related protein, Rac1, is not. In normal breast cells, Rac3 and Rac1 are turned on by hormones and by proteins called growth factors that come from outside the cells. However, since Rac3 is consistently turned on in tumors, it may be tricking cells into growing at inappropriate times. Rac3 does not have mutations, so Dr. Knaus is looking at alternative ways that Rac3's normal function can be altered. The possibilities include Rac3 changing location within the cell and Rac3 interacting with other proteins to stimulate cells to grow and spread.
Searching the Unknown: Novel Breast Cancer Genes
DNA Packaging Defects in Breast Cancer. Terumi Kohwi-Shigematsu, Ph.D., from the Lawrence Berkeley National Laboratory, Berkeley is continuing research developed with prior BCRP funding to study how chromosomal DNA is attached to proteins in the cell nucleus. This attachment provides structure to the long, looping DNA molecule. Dr. Kohwi-Shigematsu will explore the role of a protein complex, previously thought to be involved in DNA repair, as a key player in changes in DNA function in breast cancer.
Metastasis Suppressor Genes for Breast Cancer. Stanley Cohen, M.D., at Stanford University-School of Medicine, Palo Alto will attempt to discover novel tumor suppressor genes, which might be a means to inhibit breast cancer growth and progression. He is looking for a gene that, when de-activated, turns on the genetic changes that lead to breast cancer spread. He will use a novel cloning method and DNA microarrays, a technique that allows testing a tissue sample for many genes at once. When he finds a possible suppressor gene, he will check to see if it is missing in breast cancer cells, then test to see if inserting it in breast cancer cells makes them behave more like normal cells.
Suppressor Genes of Breast Cancer. Shi Huang, Ph.D., at The Burnham Institute, La Jolla will test whether three genes, and the proteins they produce that regulate the activity of genes, PFM4, PFM7, and PFM11, are suppressors of breast cancer.
A Novel Antigen Associated with Breast Cancer Metastasis. Monoclonal antibodies can be made in the laboratory; they bond with a single protein on breast cancer cells. The proteins antibodies bond with are called antigens. Jacqueline Testa, Ph.D., from the Sidney Kimmel Cancer Center, San Diego has made a monoclonal antibody called mAb 41-2 that recognizes a breast cancer antigen and blocks the spread of cancer cells. Dr. Testa is planning to characterize the structure and precise function of this newly discovered antigen in the hopes that it will eventually serve either as a biomarker (a substance present in tumors that helps physicians determine the most effective treatment) or as a therapeutic target.
Tumor Suppression by Dystroglycan in Breast Epithelial Cells. Dystroglycan is a protein found on the surface of breast epithelial cells that allows the cells to attach to a protein called laminin outside the cells. It appears that dystroglycan is either absent or not working in breast cancer cells. John Muschler, Ph.D., from the Lawrence Berkeley National Laboratory, Berkeley will study whether introducing dystroglycan into breast cancer cells changes them into more normal cells. He also plans to test variant forms of dystroglycan to discover which parts of dystroglycan's chemical structure are responsible for this action.
Analysis of a New Human Caspase in Breast Cancer. Caspases are proteins that split other proteins, causing the normal process of cell death (apoptosis). Each caspase may link a particular cell death stimulus--such as radiation, chemotherapy, or the body's immune system--to a specific series of chemical changes in the cell death process. Sug Hyung Lee, M.D., Ph.D., at The Burnham Institute, La Jolla will clone and study a new human caspase that was initially discovered in mice.
Unraveling the Path to Breast Cancer: Tumor Progression
Role of p53 in Irradiated Stroma and Mammary Carcinogenesis. By the time a breast tumor can be detected, it has developed for up to 10 years and its genes have changed to allow it to escape normal control processes. The slow progression from normal cell through the pre-cancerous and early cancerous stages is not well understood. Mary Helen Barcellos-Hoff, Ph.D., at the Lawrence Berkeley National Laboratory, Berkeley focuses on this topic, as do the two studies discussed immediately below. Ionizing radiation such as x-rays can cause cancerous changes in breast cells. Most studies concentrate on the genetic changes in epithelial cells, but radiation may change other cell types in the breast. The p53 gene tends to be mutated by radiation and is also mutated in many breast tumors. Dr. Barcellos-Hoff is investigating whether radiation causes p53 mutations in the stromal cells that are part of the framework that supports breast epithelial cells.
A Study of the Molecular Heterogeneity of LCIS. Women who have the breast disease lobular carcinoma in situ (LCIS) have an increased breast cancer risk. However LCIS may actually be several diseases, and only a subset of them may lead to a high risk for breast cancer. Sanford Barsky, M.D., from the University of California, Los Angeles will use molecular analysis and statistical analyses to correlate breast cancer risk with the different types of LCIS. v Immortalization of Human Mammary Epithelial Cells by ZNF217. The ZNF217 gene is present at higher than normal levels in many breast tumor cells, and it allows cells to continue dividing past their normal limits. Paul Yaswen, Ph.D., at the Lawrence Berkeley National Laboratory, Berkeley will investigate how ZNF217 affects proteins that suppress tumors, cell growth control processes, cell genes, and the internal skeleton of cells.
