Pathogenesis: Understanding the Disease

The Pathogenesis priority issue addresses the topic of breast tumor cell biology. An increased understanding of the initial development and progression of breast cancer at the molecular level lays the groundwork for new approaches and therapies to treat the disease.

We have organized the following descriptions into five sub-topics:

Pathogenesis research generally employs the modern tools of molecular biology to understand the unique genes and protein interactions that allow breast cancer cells to grow, become more aggressive, and spread in the body. This is referred to as ‘basic science,’ since the approach to the disease is at a very fundamental level. This allows scientists to integrate information from other cancer types, general cell biology, animal and lower organism model systems, and immunology to address research questions specific to breast cancer.

The specific impact of these research projects for women with breast cancer is not immediate. The development of basic science discoveries in a translational direction is addressed in another BCRP priority issue, Innovative Treatments. The eventual progression of new treatment approaches and therapeutics through the laboratory, pre-clinical phase, and clinical trials can take 10—15 years. The total cost of such commercial development can reach several hundred million dollars. Despite the often tangential connection of basic science to new drugs and treatments, it is well accepted that the initial research phases must occur in simple cell and animal models of breast cancer. And, the first step is the discovery of new breast cancer-related genes and proteins of interest. At each step in the drug and therapeutic development stage, hundreds of promising leads become discarded, and only a handful move on for further study. The BCRP cannot predict which of the basic science projects we fund will ultimately impact breast cancer. However, in the first five years (1995—1999) we have seen a rapid evolution of BCRP-funded basic science projects into topics of greater specificity to breast cancer. To move basic science in a direction more specific to breast cancer, the BCRP began, in 1998, to rate all applications on their potential for ‘impact’ on breast cancer. This emphasis supports “the mission of the BCRP to reduce the impact of breast cancer in California by supporting research on breast cancer, facilitating the dissemination of research findings, and their translation of research into public health practice.”

Unfortunately, each new gene, protein, and biochemical discovery is hailed as the ‘next cure’ for cancer. Over two years ago a famous Nobel Laureate predicted in the New York Times that the discovery of the angiogenesis inhibitors, endostatin and angiostatin, would be the cure for cancer—within two years! Such optimism leads the public to become sceptical, or even cynical, about the claims for basic science discoveries. These opinions notwithstanding, the underlying creative process to prevent and cure breast cancer is stimulated by two primary factors. First, new young investigators must be supported to start careers in breast cancer research. Secondly, research that uses innovative approaches and challenges the current dogma must be supported.

Research Conclusions

Outbreak—how cancer spreads: angiogenesis, invasion, and metastasis

Pierre Desprez, Ph.D. of the California Pacific Medical Center completed a New Investigator Award entitled “The Invasive Nature of Epithelial Breast Cancer Cells,” which looks at the role of a protein called Id-1 in breast cancer metastasis. Id-1 binds to specific regions of genes to regulate their tissue-specific production in many cell and cancer types. Dr. Desprez found that the presence of Id-1 is associated with breast tumors that invade their surrounding environment. He also found that when Id-1 is introduced into breast cells that are not invasive, they become invasive. Finally, the production of Id-1 is regulated by certain hormones. For example, estrogen causes the cell to make more Id-1, whereas progesterone decreases Id-1 production. These results indicate that regulation of Id-1 could be one of the important factors for determining the aggressiveness of breast cancer. Findings from this project were published in the journal Molecular and Cellular Biology (Aug 1998; 18(8): 4577-88).

David Rose, Ph.D., D.V.M. at The Scripps Research Institute, completed a 2-year Postdoctoral Fellowship on Integrin Receptor Activation in Breast Cancer. He examined proteins on the surface of breast cancer cells that are critical to cell migration. This tumor cell receptor, an integrin called a4b1, binds to another receptor on cells lining blood vessels, called Vascular Cell Adhesion Molecule (VCAM)-1. Mutant cell lines were developed that had altered a4b1 binding affinity and showed increased migration. It appears that a4b1 is sensitive to the intracellular signaling pathways known to be changed in breast cancer cells. Future work could reveal how a4b1 is directly associated with breast cell migration. In addition, soluble inhibitors of a4b1 adhesion could block cell movement.

Alex Strongin, Ph.D. at the La Jolla Institute of Experimental Medicine completed a Research Project award entitled “How are Collagenases Involved in Breast Cancer Metastasis?” A group of enzymes called collagenases are able to dissolve the scaffolding supporting the breast cells (extracellular matrix), thus performing a necessary step for allowing cancer cells to move from the breast to other parts of the body. Dr. Strongin is delineating the critical steps in this process and identifying the key players. He found that the activation of an enzyme called MMP-2 depends on the activity of two other proteins, MT1-MMP (a protein present in most aggressive breast tumors) and the avb3 integrin receptor. He also found that MMP-2 must be attached to the membrane of the cell in order for it to affect cell movement, cell shape and localized digestion of the extracellular matrix. Several articles were published during this project, most recently in the journal Molecular and Cellular Biology (Nov 1999; 17(11): 6598- 608).

Karin Zeh, Ph.D. at The Burnham Institute began project on Cell Adhesion Signaling Defects in Breast Cancer was focused on investigating a gene in breast cells, called plakoglobin, that is involved in cell-cell association events to maintain the normal epithelial structure. She was developing a technology of gene knockout (i.e., gene deletion) that would be specific to the mammary gland in adult mice. She completed the initial stages of the project to develop an indicator mouse strain having the appropriate expression of a marker gene (b-galactosidase). She resigned her Fellowship after one year to pursue a career in biotechnology.

Too much cell growth: defective messages and internal signaling

Myles Cabot, Ph.D. of the John Wayne Institute for Cancer Treatment completed a project entitled “Nongenomic Actions of Antiestrogen,” in which he investigates the mechanism of action of agents such as tamoxifen on the components of the cellular membrane. Tamoxifen action can occur through the membrane by causing changes in the elements of the cellular membrane called second messengers. Dr. Cabot found that one of these second messengers, called PKC-epsilon, is specifically activated by tamoxifen. This pathway could be the one operating in breast cells that are estrogen receptor negative but responsive to tamoxifen. Additionally, he found that one of the actions of tamoxifen is to block glycolipid metabolism, a biochemical pathway used by the cancer cells to resist Adriamycin therapy, thus supporting the observation that tamoxifen can reverse resistance to chemotherapy. This research produced several publications, including ones in the International Journal of Cancer (Mar 4, 1997; 70(5): 567-74 and Sep 11, 1998; 77(6): 928-32). This initial work supported by BCRP allowed Dr. Cabot to obtain federal funding to continue research in this area.

Shiuan Chen, Ph.D. of the Beckman Research Institute of the City of Hope completed a project entitled “Control of Estrogen Production in Breast Cancer.” He studied how estrogen is produced in fat tissues and found that cancer-free areas of the breast produce less estrogen than cancerous areas. He also found that the regulation of estrogen production by the aromatase gene was under the control of hormones in cancer-free areas, whereas a protein called cAMP regulated estrogen production in areas with cancer. Based on the analysis of the different regulatory areas of the aromatase gene, Dr. Chen hypothesizes that when cancer cells are exposed to estrogen, they produce cAMP and then the control of estrogen synthesis switches from a hormone-dependent to a cAMP-dependent process. This study may reveal a fundamental difference between benign tissue and cancerous tissue. Dr. Chen reviewed this field in the journal Frontiers in Bioscience (Aug 6, 1998; 3:922-33).

Michael Karin, Ph.D. at the University of California, San Diego completed a 3-year project on Regaining Control over Breast Cancer Cells. The aim was to study how breast cancer cells can survive in the presence of signals (e.g., chemotherapy and radiation) that trigger the process of apoptosis, which is programmed cell death. They found that breast cancer cells could escape death by activating the gene regulatory protein (transcription factor) called NF-kB. This research was published in Cell (Nov 1 1996; 87(3): 565-76). Inhibitors of NF-kB and its associated proteins can now be investigated as potential inhibitors of breast cancer in animal models. This approach could render breast cancer cells more sensitive to cell death caused by either chemotherapy or immune attack.

Juan Zapata, Ph.D. from The Burnham Institute was funded for a 2-year Postdoctoral Fellowship to study TRAF-regulated Signal Transduction in Breast Cancer. This project is related to Dr. Karin's project described above. In this project, Dr. Zapata studied the pathways by which apoptosis (cell death) signals are communicated within breast cancer cells. Comparing normal breast tissue and breast cancer by microscopy techniques, he found that the amounts of a protein called TRAF-4 was decreased. In further studies, he showed that mutating the cell surface receptor (CD40) for TRAF signaling would still allow the relay of other signals through alternate cell death pathways via NF-kB (see Dr. Karin's project above). Thus, breast cancer cell death appears to be regulated by both positive (TRAF) and negative (NF-kB) apoptosis factors. These studies were recently published in the Journal of Biological Chemistry (Aug 6, 1999; 274(32): 22414-22).

Sahn-Ho Kim, Ph.D. from the Lawrence Berkeley National Laboratory was funded for 2 years as a Postdoctoral Fellow to study Loss of Tumor Suppressor Proteins in Breast Cancer Cells. His initial plan to find proteins that interact with the retinoblastoma (Rb) tumor suppressor was unsuccessful. He then redirected his project to discover proteins that interact with telomeres, which are the ends of chromosomes and are critical to controlling the capacity for cells to divide indefinitely. Using the yeast two-hybrid system, he identified a telomere-associated protein called Tin2. The relationship of Tin2 to breast cancer is not yet established, but this new information could explain the capacity of cancer cells to escape the normal cell aging process.

Cary Lai, Ph.D. of The Scripps Research Institute completed a project entitled The Role of a Newly Discovered Neuregulin in Breast Cancer. This one-year award explored the possibility that a newly discovered molecule, named NRG-4, could belong to a family of genes called the neuregulins. Members of the neuregulin family are ligands (they bind to a receptor and activate it) for the ErbB family of receptors. ErbBs, particularly ErbB2 (also called Her-2/neu), have proven to play important roles in the development and treatment of breast cancer. Dr. Lai found that the NRG-4 qualifies as a member of the neuregulin family because it activates ErbB4, although it does not activate ErbB2. The identification and characterization of NRG-4 provides a more complete picture of the factors that influence treatments such as heregulin-based therapies.

Ichiro Maruyama, Ph.D. of The Scripps Research Institute completed a project entitled Growth Factor Receptor Activation in Breast Cancer, investigating how the epidermal growth factor receptor (EGFR) becomes active. The current thinking is that two molecules of EGFR come together to form the active structure (a dimer). However, by creating mutant EGFR components, Dr. Maruyama was able to show that the dimer structure can form without bound EGF. Thus, dimer formation is not sufficient for EGFR activation, but rather it is the relative orientation of two molecules in its dimer structure that is needed. This information will ultimately be helpful for guiding future research into targeting EGF-like receptors.

Alexandre Nesterov, Ph.D. of the University of California, San Diego completed a Postdoctoral Fellowship entitled “Molecular Mechanisms of EGF Receptor Endocytosis.” The goal of the research was to lay the groundwork for developing an approach to therapy that specifically targeted epidermal growth factor (EGF) - a growth hormone involved in breast cancer. The approach is based on the observation that the receptor for epidermal growth factor (EGFR) is brought inside the cell (endocytosis) from the cell surface and degraded in order to inactivate it. If this process could be accelerated specifically for EGF-like receptors in tumor cells, this could serve as a way to control tumor growth. Dr. Nesterov was able to identify a previously unknown protein called ‘Catnip’ that may be involved in this process. He was also able to develop a method that is being used to identify previously unknown signaling components of the epidermal growth factor receptor.

Kevin Sato, Ph.D. at The Scripps Research Institute finished a 2-year Postdoctoral Fellowship to investigate Disruption of Cyclin E/Cell Cycle in Breast Cancer Cells. The ability of cells to divide is regulated by proteins called cyclins, which are the checkpoints in the cell cycle. One of these checkpoint proteins, called Cyclin E, appears elevated in breast cancer and is associated with a partner protein, cdk2. Dr. Sato's project used both antisense (inhibitory DNA fragments) and knockout (gene deletion) approaches to reduce cyclin E amounts in cells. However, these cyclin E blocking approaches led to only modest increases in cell division times. Interestingly, in the course of these studies, the presence of a novel inhibitor of cdk2 was demonstrated. Thus, breast cancer cells that were inhibited for cyclin E possessed an unexpected ability to maintain adequate levels of cyclin E/cdk2 complex by an alternate process.

Michael Stallcup, Ph.D. of the University of Southern California has completed a project entitled Estrogen Receptor-Interacting Proteins in Breast Cancer. The goal of this project was to characterize some previously unknown proteins that could potentially be interacting with the estrogen receptor (ER) and determine whether they could regulate estrogen action by interfering with the function of the receptor. Dr. Stallcup found that the novel protein fragments he originally identified (GRIP2 and GRIP3) were not natural genes; however, he did find a portion of the natural gene GRIPI that could preferentially block the necessary step of the estrogen receptor binding to its coactivator proteins. He found that GRIPI interferes with the estrogen receptor function by binding to multiple regions along the estrogen receptor. During this investigation he also discovered a novel estrogen receptor-related protein named estrogen receptor related protein 3, or ERR3, that can turn on some of the same genes as the estrogen receptor, but does not require estrogen to do so. If breast cancer cells are found to have too much ERR3, it would lend credence to the possibility that this protein is responsible for estrogen independent growth. Dr. Stallcup published work from this project in several papers, most recently in the Journal of Biological Chemistry (Aug 6, 1999; 274(32): 22618-26).

Mistakes on the master blueprint: molecular genetics and gene regulation

Robert Oshima, Ph.D. from The Burnham Institute completed a project to study Oncogene Regulation in Mammary Cancer. The focus of his research was the gene regulatory protein (transcription factor) called Ets2. Dr. Oshima tried several techniques to reduce the amounts and block the activity of this protein. Using human mammary tumors grown in mice, he found that decreasing the amount of Ets2 in cells by half resulted in much smaller tumors. These results were published in Cancer Research (Sep 1,1999; 59(17): 4242-6). Many important breast tumor genes are believed to be regulated by Ets2, and further work will be needed to show how specific reductions in this protein might inhibit tumor formation.

Philippe Pujuguet, Ph.D. from the Lawrence Berkeley National Laboratory completed a 2-year Postdoctoral Fellowship to investigate ECM-Regulated Transcription in Breast Cancer. The idea underlying this research was that the material that surrounds cells (the extracellular matrix, or ECM) interacts with breast cells to either prevent or promote their transformation into a malignant state. To study this process, Dr. Pujuguet examined how production of an important milk protein, casein, was regulated by factors (called transcription factors) that control genes. This work led him to discover that a particular enzyme that acts on histones (chromosome binding proteins) appeared to be directly associated with the presence or absence of the extracellular matrix. These findings help explain how normal breast cells maintain their morphology and synthesize proteins associated with the non-transformed state. This project was published in the journal Molecular and Cellular Biology (Apr 1998; 18(4): 2184-95).

David Zarling, Ph.D. at the Pangene Corporation was funded for a 2-year IDEA project to study Recombination and Mutation in Breast Cancer. He investigated how breast cancer cells can recover from DNA damage induced by either radiation or chemotherapeutic drugs. He found that a DNA recombination and repair protein, called Rad51, was induced in breast cancer cells in response to radiation. Inhibition of Rad51 by both genetic knock-outs and using novel, proprietary inhibitors developed at Pangene would make the cells much more sensitive to radiation treatment. Thus, use of these new inhibitors could increase the effectiveness of radiation therapy.

Searching the unknown: novel breast cancer genes

Terumi Kohwi-Shigematsu, Ph.D. from the Lawrence Berkeley National Laboratory completed a 3-year project to study The Role of MAR-Binding Protein in Breast Cancer. She examined the process and the proteins involved in the attachment of chromosomal DNA to the skeletal framework of the cells' nucleus. Unexpectedly, she found that a previously described DNA repair protein, called poly ADP-ribose polymerase (PARP), is present in high amounts in breast cancer and participates in chromosomal DNA attachment. This is important, because previously it was believed that PARP and its companion proteins only bound to DNA nicks associated with DNA damage. The potential role of the PARP system in breast cancer was confirmed when experiments to reduce PARP levels in breast cancer cells lines resulted in a loss of invasiveness and ability to form tumors in mice. The most recent findings for this project were published in the Journal of Biological Chemistry (Jul 16, 1999; 274(29): 20521-8).

Claudia Lin, Ph.D. at the Lawrence Berkeley National Laboratory completed a 2-year Postdoctoral Fellowship to work on a Targeted Search for New Transcription Factors in Breast Cells. The overall goal was to discover how gene regulation becomes altered during the differentiation of breast cells. Previously, she had described the Id-1 transcription factor, which is being investigated by other BCRP funded projects awarded to Dr. Pierre Desprez. In Dr. Lin's research, the yeast two-hybrid method was used to find a protein binding partner of Id-1, which is called ITF-2. She found that ITF-2 binding activity towards DNA would increase significantly upon growth arrest, and that artificially increasing amounts of ITF-2 within breast cells would cause morphological changes and b-casein milk protein production. The significance of this work is that it leads to strategies to understand the normal function of breast cells, which could be maintained to prevent the development of breast cancer.

Michael Press, M.D., Ph.D. of the University of Southern California has completed a project entitled Genes Which Cause Increased Cellular DNA in Breast Cancer. Dr. Press was looking for the human gene equivalents to ‘rum1’, a yeast gene that regulates DNA. He found two DNA fragments in human cells that qualify as human counterparts to rum1, which turned out to be different parts of the same gene. The protein product of the gene could be found in all of the adult tissues and was found at similar levels in tumor tissues. There were also no mutations in the gene in any breast cancer cell line. Therefore, although this project successfully identified a human cousin to the yeast rum1 gene, it does not appear that this gene plays a role in breast cancer development.

Mathias Treier, Ph.D. at the University of California, San Diego completed a 2-year Postdoctoral Fellowship on Potential Tumor Suppressors in Breast Cancer. This project also investigated a gene regulation process that could be more ‘global’ in nature. He focused on a protein that controls gene activity called the nuclear receptor corepressor (N-CoR). The importance to breast cancer is that the N-CoR is thought to form protein complexes that modulate the estrogen receptor. Thus, any defects in the N-CoR could lead to resistance to tamoxifen therapy. Dr. Treier attempted to generate mice that lacked the N-CoR, but these animals would not survive beyond the embryonic stage. He plans to investigate this problem by surgically transplanting tissue from embryonic N-CoR-deficient mice into normal mice and allowing the genetically deficient mammary tissue to develop in a so-called ‘chimeric’ state. In other planned work, the role of the N-CoR will be studied in cell models.

Fabiana Guerra-Vladusic, Ph.D. of the Lawrence Berkeley National Laboratory received a Postdoctoral Fellowship entitled Characterization of Heregulin Targeted Genes. She was able to make significant progress in her research project, which characterized the cellular and genetic effects of heregulin in breast cells. Heregulin is a molecule that binds to ErbB2 (also called Her-2/neu), a receptor that is an important protein in the development and treatment of breast cancer. Dr. Guerra-Vladusic defined the genes and proteins present when heregulin production is constantly turned on in the cell. She found that this caused the breast cells to stop dividing, increase in size and begin to die. She also found that these cells produced less ErbB2 and ErbB3, but more of a nuclear localized protein called PEA3. This observation opens up the therapeutic possibility that cells with too much ErbB2 can be induced to die by stimulating PEA3 production or activity. This research was recently published in the International Journal of Oncology (Nov 1999; 15(5): 883-92). Dr. Guerra-Vladusic resigned her fellowship to work in a biotechnology company.

Sergei Malkhosyan, Ph.D. at The Burnham Institute completed an IDEA project on Molecular Karyotyping of Breast Cancer by DNA Fingerprinting. He developed a novel approach to detecting genetic changes associated with breast cancer, called Arbitrarily-Primed PCR (AP-PCR). About 80 different breast tumors were analyzed and the gain or loss of chromosomal regions was tallied. Gain of chromosomal segments was characteristic of chromosomes 1, 4, 8, and 10. Loss of chromosomal material was often associated with chromosomes 4, 6, 9, 10, 11, and X. The goal is localize the gains and losses to more specific regions and correlate these with already identified breast cancer genes.

Unraveling the path to breast cancer: tumor progression

Anissa Agadir, Ph.D. of The Burnham Institute completed a Postdoctoral Fellowship entitled Anti-Breast Cancer Activity of Vitamin A Derivatives. Retinoids are Vitamin A derivative compounds that have been investigated for their cancer preventative properties. In order to optimize their usage, it is important to understand the biology of cellular functions. Retinoids are known to turn on different classes of receptors including the RAR class and the RXR class. Retinoids that operate through different families may have different biological consequences. Dr. Agadir used synthetic retinoids that specifically activate the different classes of receptors and found that the RXR pathway can cause cell growth inhibition when the RAR pathway fails. She also found that effectiveness of retinoids in blocking the TPA (a tumor promoter) pathway may predict the success of retinoid treatment in breast cancer patients based on the RAR status. Her results indicate that synthetic retinoids that effectively and specifically block TPA-induced cell transformation are promising candidates for further study. Several publications resulted from this project, including one in the journal of Molecular and Cellular Biology (Nov 1997; 17(11): 6598-608).

Chin-Shwun Lin, Ph.D. at the University of California, San Francisco finished a 3-year project to study the Role of L-Plastin in Breast Cancer. The plastins are a small family of proteins that associate with the internal cytoskeleton of cells. Normal breast cells do not contain L-plastin, but breast cancer cells commonly express this protein. Dr. Lin first developed evidence showing that L-plastin can be measured in breast tumor samples using antibodies, and he showed that this protein could be a useful diagnostic marker. Secondly, he found that L-plastin was found inside breast cells at the leading edge of the cell, which indicated a role in cell invasion. This hypothesis was confirmed by introducing L-plastin into low-grade breast cancer cell lines and showing an increase in invasive properties. Thirdly, Dr. Lin found that the L-plastin gene contained estrogen responsive regions, but it could also be regulated by androgens. Work from this project was published the journal DNA and Cell Biology (Dec 1998; 17(12): 1041-6).

Research in Progress

Outbreak—how cancer spreads: angiogenesis, invasion, and metastasis

A sampling of BCRP-funded research grants in progress under this sub-topic illustrates a diversity of approaches. These include the searching for novel motility proteins, understanding complex regulatory pathways, and discovering how tumors can grow in the body and still evade immune detection. Key players in breast cancer cell movement are the tumor-secreted proteases that digest the material that surrounds cells (the extracellular matrix). Pierre Desprez, Ph.D. at the California Pacific Medical Center, is continuing the search for a novel breast cancer metalloproteinase (MMP) that appears to be induced by the gene regulatory protein, called Id-1. Since these proteases are secreted from cells, they could represent a novel target for early detection by their presence in blood or urine. Next, Ulla Knaus, Ph.D. at The Scripps Research Institute is continuing a project to determine how an intracellular signaling pathway in breast cancer cells is associated with cell migration. She is introducing mutant forms of signaling proteins, called Rac, into breast cancer cell lines and measuring changes in the cytoskeleton and cell growth. In addition to cell movement, this project is also examining the cell cycle and other signaling pathways. Finally, an emerging field of investigation in cancer research has received considerable attention during the past two years. This is the process of angiogenesis, which is the growth of new blood vessels into tumors that allow them to increase in size beyond 1-2 mm and spread in the body. Pragada Sriramarao, Ph.D. from the La Jolla Institute of Experimental Medicine is continuing a project focused on the vascular adhesion processes of the blood circulation within tumors. He uses small tumor spheroids that, when implanted into mice, can be visualized by special microscopy. He studies the immune regulatory factors (e.g., TNF-beta, IL-4) that allow circulating white blood cells to arrest in the tumor microcirculation. How the tumor microcirculation develops, and how it differs from the microcirculation of normal tissues is an area of active research.

Too much cell growth: defective messages and internal signaling

Several projects are progressing in this topic area. First, Kathryn Ely, Ph.D. from The Burnham Institute is funded to investigate the 3-dimensional structure of the tumor suppressor retinoblastoma (Rb) protein and its binding partner protein, called RIZ. If she can locate the critical interaction regions of these two proteins, then future work could translate these findings into drug development. Other tumor suppressors, such as p53, protect the body from unregulated cell growth, since they can trigger cell death following DNA damage by either radiation or chemotherapeutics. Glenn Rosen, M.D. at Stanford University is investigating the action of a novel drug called PG490, which appears to sensitize breast cancer cells to the actions of tumor necrosis factor (TNF). This immune factor can trigger cell death. His findings extend the work of Drs. Karin and Zapata (described above) by supporting the role of a gene regulatory protein, NF-kB, as a key player in allowing breast cancer cells to avoid apoptosis (i.e., cell death). Apparently, PG490 interferes with the protein pathway that links TNF and NF-kB. Dr. Rosen's research was recently published in the Journal of Biological Chemistry (May 7 1999; 274(19): 13451-5). Finally, Koji Itahana, Ph.D. from the Lawrence Berkeley National Laboratory is examining a novel aspect of p53 mutations in breast cancer cells. It is known that many breast cancers are associated with mutations in p53. It has been assumed that these p53 mutations serve to inactivate the protein function. Challenging this paradigm, Dr. Itahana is looking for p53 mutations that have a ‘gain-of-function’ activity.

Mistakes on the master blueprint: molecular genetics and gene regulation

Shu-ichi Matsuzawa, Ph.D. from The Burnham Institute is continuing to study a human homologue of a fruit fly growth arrest protein, called Siah. He has found that breast cancer cells contain all three known members of the Siah protein family. Further work will show how other proteins bind to and regulate Siah activity, and whether changes in Siah amounts will affect breast cell proliferation. Heinz Ruffner, Ph.D., at The Salk Institute for Biological Studies has found specific sites where the BRCA-1 (breast cancer-1) protein is phosphorylated. These studies will help resolve the issue of how BRCA-1 is associated with known cell regulation pathways inside cells, and how mutations in BRCA-1 can lead to increased risk for developing breast cancer. Dr. Ruffner recently published his findings in the journal Molecular and Cellular Biology (Jul 1999; 19(7): 4843-54).

Searching the unknown: novel breast cancer genes

Donna Albertson, Ph.D. at the University of California, San Francisco is continuing a project to map and identify critical breast cancer oncogenes (i.e., cancer-inducing genes) on human chromosome 20. She has identified a potentially important mammary oncogene, called ZNF217. Her results were published in the Proceedings of the National Academy of Sciences, USA (Jul 21, 1998; 95(15): 8703-8) and Nature Genetics (Oct 1998; 20(2): 207-11). Sanjeev Galande, Ph.D. at the Lawrence Berkeley National Laboratory is continuing a Postdoctoral Fellowship with Dr. Kohwi-Shigematsu (see above) to study the chromosomal DNA attachment process in the nucleus. It appears that a type of DNA repair mechanism in normal cells becomes altered in breast cancer, and this causes abnormal gene expression and cellular functions. Fumiichiro Yamamoto, Ph.D. of The Burnham Institute is continuing his search for previously unknown genes that may be playing a role in breast cancer. He is analyzing the pattern of genes that are turned on or off by examining changes in DNA methylation. Finally, Paul Kaufman, Ph.D., at the Lawrence Berkeley National Laboratory is funded to study cellular aging and its relationship to proteins that normally protect chromosomes. He is working on a protein called CAF-1 (chromatin assembly factor-1). Most breast cancers occur in older women, so understanding aging at the cellular and molecular level is critical in devising strategies to prevent the very early stages of cancer progression.

Unraveling the path to breast cancer: tumor progression

Older women are at greater risk for developing breast cancer. How is this increased risk associated with specific cellular and genetic changes? Dr. Judith Campisi, Ph.D. at the Lawrence Berkeley National Laboratory is continuing a project to understand how the connective tissue cells, called fibroblasts, become altered with age and contribute to localized changes to permit the neighboring breast cells to become altered. Her specific interest is the molecular communication between these two cell types that involves the secretion of a growth factor (heregulin) by the fibroblasts, and the stimulation of its receptor (Her-2) on breast cells. More information on the local environment of breast cells is critical to understanding the changes leading to breast cancer.

Normal cells have limits placed on their ability to divide; the acquisition of cell immortality is a critical step in tumor progression. Martha Stampfer, Ph.D. of Lawrence Berkeley National Laboratory is using a cultured human mammary epithelial cell (HMEC) system and microarray technology to identify those genes whose products are altered once cell become immortal. She has identified 12 genes thus far and will perform additional experiments to characterize them. As tumors become more aggressive, they begin to look and behave like immature breast cells. G. Shyamala, Ph.D. also at the Lawrence Berkeley National Laboratory is investigating the role of hormones, such as progesterone, in maintaining the maturity of breast cells. She is finding these hormones play an important role in maintaining the integrity of the surface (i.e., basement membrane) that supports the breast cells in their normal functions.

Recently Initiated Research

In 1999, the BCRP funded 18 new grants focused on breast cancer Pathogenesis. The adhesion, migration, and spread of breast cancer cells in the body continue to be areas of intense research interest. Many of these new projects focus on angiogenesis, the development of the tumor blood supply necessary for growth and metastasis. A major clinical advantage to targeting angiogenesis is the ability to attack secondary sites of tumor growth. Jan Schnitzer, M.D. at the Sidney Kimmel Cancer Center is funded to investigate the unique surface proteins on the endothelial cells that line blood vessels within tumors. The discovery of unique proteins in blood vessels that feed tumors might provide targets to selectively attack breast cancer and spare the normal tissues of the body. Kristiina Vuori, Ph.D., from The Burnham Institute is studying a possible mechanism of action of a new anti-angiogenic drug, endostatin, which is believed to interact with a cell adhesion receptor. The major endothelial and breast tumor integrin, avb3, is the subject of two projects. First, an endothelial proteinase is being studied for its binding to this receptor by YingQing Sun, Ph.D. at The Burnham Institute. This interaction would serve to facilitate endothelial migration during angiogenesis, and could be the basis for new therapeutics, if confirmed. Secondly, Alex Strongin, Ph.D. at The Burnham Institute is studying this same protease-integrin interaction, but in breast cancer cells, in order to explain how cancer cells are capable of invasion and to explore new approaches to block this movement. Brett Premack, Ph.D. from the University of California, Los Angeles is surveying breast cancer cells for the presence of chemokines and chemokine receptors. These receptors are important for leukocyte (white blood cell) motility and invasion and, if found on breast cancer, could be functioning in a similar manner. Breast cancer cells travel to distant sites via the blood and lymph systems. Brunhilde Felding-Habermann, Ph.D. from The Scripps Research Institute is investigating the attachment of breast cancer cells to the endothelium by the combined function of the integrin avb3 on the cancer cells and another receptor, ICAM-1, on the endothelial cell. A key aspect of this research is to isolate and study circulating breast cancer cells from the blood of experimental animals. Finally, Sanford Barsky, M.D. from the University of California, Los Angeles has a unique model system in mice to study inflammatory breast carcinoma, which spreads locally in the breast via the lymph. He is investigating how this rare form of breast cancer differs from more common types of the disease.

Newly funded research under the topic of breast cancer cell growth regulation focuses both on the intracellular regulatory systems linked to growth factors and how breast cells evade signals that cause cell death (apoptosis) for normal cells. Elena Pasquale, Ph.D. at The Burnham Institute is funded to study a new breast cancer messenger protein within cells for the ability to contribute to growth, metastasis, and tumor progression. The oncogene growth receptor Her-2 is present in about 30% of cancers, and is associated with a poor prognosis. Although the new therapeutic Herceptin was released last year, this topic is of continued research interest. Janis Jackson, M.D. at The Scripps Research Institute is investigating how Her-2 and related growth receptors signal through various signaling kinases, the Ras oncogene family. Ronald Weigel, M.D., Ph.D. from Stanford University is studying how gene regulation proteins (i.e., transcription factors) serve to mediate the estrogen response. Such proteins could be become surrogate markers to evaluate the effectiveness of estrogen receptor-based therapies (e.g., tamoxifen and raloxifene). New aspirin-like drugs offer interesting new avenues to treat cancer. Youngsoo Kim, Ph.D. at The Burnham Institute is focused on the role of COX-2, a target of aspirin-like drugs, for its presence in breast cancer and association with apoptosis pathways. Finally, Heimo Strohmaier, Ph.D. at The Scripps Research Institute is funded to study the degradation of a cell cycle regulation protein. A failure to regulate the amount of this protein in breast cancer could lead to excess cell growth by permitting cells to pass through cell cycle checkpoints in an uncontrolled manner.

It is well accepted that genetic differences, both subtle and dramatic, underlie the initiation and progression of breast cancer. Two newly-funded projects are examining genes involved in breast cancer growth. First, Devon Thompson, Ph.D. from Stanford University has found a new gene, called hAG-2, that appears to associate with the estrogen receptor and could underlie resistance to anti-estrogen therapy. Secondly, Katherine Ely, Ph.D. at The Burnham Institute is conducting structural analysis of an apoptosis-associated gene, BAG-1, for its indirect association with the estrogen receptor, which also could be involved in anti-estrogen resistance. Genetic repair and recombination within and between different chromosomes lead to severe genetic defects that characterize advanced forms of breast cancer. Joanna Albala, Ph.D., working at the Lawrence Livermore National Laboratory has discovered and is characterizing a new chromosome recombination gene, RAD51B, which appears to be capable of associating with the hereditary breast cancer genes, BRCA1 and BRCA2. DNA damage from environmental causes, such as radiation and chemical mutagens, must be repaired correctly for cells to remain normal. Thus, there is interest in how acquired defects in normal DNA repair systems could lead to breast cancer. Eric Brown, Ph.D. from the California Institute of Technology is studying a potential DNA damage repair gene, called ATR, for its role in breast cancer and its association with BRCA1 and the tumor suppressor, p53. Progression of breast cancer involves processes of differentiation, which are permanent cell changes in gene expression and phenotype. Two newly funded projects are examining how certain proteins that determine whether a gene is utilized to make protein are related to breast cancer. Bogi Andersen, M.D. at the University of California, San Diego is investigating a gene regulation protein, LMO-4, which is involved in white blood cell differentiation and is thought to be associated with breast cancer progression. Finally, Pierre Desprez, Ph.D. from the California Pacific Medical Center is funded to continue his studies on a novel gene regulation protein, called Id-1, which appears to be essential for the expression of cell invasion proteases involved in breast cancer cell migration.