Pathogenesis: Understanding the Disease
Breast cancer does not come about as a single event, but rather develops through a series of events, becoming what we diagnose as "cancer" and then gaining increasing degrees of aggressiveness and resistance. Understanding these events is critical to interpreting and intervening in the disease process. Discoveries made in this area are leading to the development of new targets and agents for the treatment of breast cancer.
Research Conclusions
The presence or absence of progesterone receptor has long been used as a predictive marker for prognosis in breast cancer, but its function in breast cancer development is still in question. Dr.G. Shyamala at the Lawrence Berkeley National Laboratory completed a one-year IDEA grant (Progesterone Action in Human Breast Cancer) to create a model that would allow researchers to explore this question. Dr. Shyamala has identified a cell line that does not form tumors but has significant levels of functional progesterone receptors. As the cells lose their progesterone receptors, they begin to behave increasingly like tumor cells. This research has provided a new model for studying the effects of the presence or absence of progesterone receptors on the generation of the tumor phenotype.
Making genetic distinctions between normal and tumor cells is an important area of investigation in breast cancer research. It is especially beneficial to be able to compare the normal cell genes to the tumor cell genes and determine which ones differ. In his one-year IDEA project New Method for Measuring Breast Cancer Gene Expression, Dr. Daniel Pinkel at the Lawrence Berkeley National Laboratory improved on a technique that examines abnormalities in several genes simultaneously. In this technique, the RNA from populations of normal cells and tumor cells is tagged with different colored fluorescent markers and allowed to compete for a matching sequence that is mounted on a microarray. The cell population (i.e. normal cells) with more of a given gene product will out-compete the cells from the other population (i.e., tumor cells) for a spot on the array. The technique developed by Dr. Pinkel requires fewer numbers of cells to detect differences than those developed by others and, therefore, expands the types of tissues that can used for comparisons.
Dr. Thorsten Heinzel of the University of California, San Diego successfully completed his two-year postdoctoral fellowship, entitled Isolation of Estrogen Receptor Co-factors from Breast Tumors. Dr. Heinzel was awarded this fellowship in order to determine the underlying mechanisms of the estrogen receptor function in the presence or absence of antiestrogen. Previous work has shown that, in order for estrogen to turn genes on or off, it must first bind its receptor. This estrogen-receptor complex must bind other proteins (cofactors) that in turn bind the appropriate DNA sequences. Dr. Heinzel identified novel cofactors SRC/N-CoA and CBP, that serve not only as mediators of estrogen function, but also as mediators for other growth factors or hormones. In addition, he identified a co-repressor, N-CoR, that can bind estrogen receptor in the presence of antagonists. Decreased levels of N-CoR correlate with the acquisition of tamoxifen resistance in mice. The interaction of novel therapeutics with these coactivators and corepressors may provide a new pre-clinical index of the value of breast cancer treatments in individual women.
In her study, Novel Ways to Control the Growth of Mammary Epithelial Cells, Dr. Susanne Koch at The Scripps Resarch Institute has been investigating the processing of the epidermal growth factor (EGF) receptor. The EGF receptor lies at the surface of the cell where it attaches to EGF, which turns on the receptor. One of the ways that a cell turns off a signal from a receptor is to bring the part of the membrane where the receptor is sitting into the cell and degrade the receptor. Dr. Koch has been studying dynamin, a protein that is integral to the process of bringing the receptor inside of the cell. Dr. Koch identified certain proteins that partnered with activated dynamin, and therefore may be involved in regulating the internalization and degradation of the EGF receptor. Dr. Koch resigned from her postdoctoral fellowship in order to take a full-time teaching position.
Dr. Qiang Liu at The Scripps Research Institute, Zinc Finger Proteins to Study Breast Cancer Angiogenesis, was funded to develop a novel class of DNA-binding proteins that target integrin avb3, which is involved in the process of blood vessel formation (angiogenesis). He designed, constructed and characterized two new DNA-binding proteins capable of specifically binding unique DNA sequences. He has shown that these DNA-binding proteins are capable of turning target genes on or off. These DNA-binding proteins are potential tools to treat breast cancer by acting as switches that can turn specific genes such as integrin avb3 on or off. Dr. Liu resigned from his postdoctoral fellowship in order to begin a research career in a biotechnology company.
Dr. Deborah Cadena at the University of California, San Diego completed a two-year New Investigator project entitled ErbB2 and Control of Growth in Breast Cancer Cells. She studied the proteins inside the cell that control the function of two critical growth receptors, ErbB2 and the epidermal growth factor (EGF) receptor. When present, both the ErbB2 and EGF receptors on the cell surface contribute to more aggressive forms of breast cancer. Dr. Cadena studied two regulatory proteins for these receptors. The first protein, SNX1, was found to decrease amounts of ErbB2 and the second, MLD, directly interacts with the growth receptors in the cell membrane also lowering receptor amounts. This research opens up new protein targets, contributing to our understanding of the growth of certain breast cancers.
Dr. Nam Woo Kim at Geron Corporation finished a two-year Research Project. In Telomerase: a Factor for Early Detection of Breast Cancer, he studied a protein that, when present, can make cancer cells immortal and gives them unlimited growth potential. This telomerase protein repairs the ends of chromosomes during cell division. Dr. Kim, in collaboration with researchers at the University of Texas, San Antonio, reported in the Journal of the National Cancer Institute (89:1874-1880, 1997) that telomerase activity is associated with more aggressive tumor behavior. He used a proprietary technology to analyze breast cancer samples with extensive biomarker and diagnostic data for possible correlation with telomerase activity. His published work strengthens the potential for telomerase as a new breast cancer biomarker or target for therapeutic intervention.
Dr. Lisa McPherson at Stanford University was successful in her two-year Postdoctoral project, Estrogen Receptor Regulation in Breast Cancer, which involved the cloning of a critical regulatory protein for the estrogen receptor (ER) gene. The estrogen regulatory factor (ERF-1) cloned in this project is a transcription factor that binds to DNA. Dr. McPherson discovered that ERF-1 is closely related to certain other transcription factors. She is now able to conduct further molecular studies to understand how the ER is regulated in breast cancer cells. More effective therapies against the ER has potential to treat breast cancers in older women.
In his study, Why do 70% of Breast Cancers Metastasize to Bone?, Dr. Jose Millan at The Burnham Institute addressed the issue of cancer cell spread in the body in a one-year IDEA project. His approach was to use a phage display peptide library to discover new receptors on breast cancer cells that could be potential sites for attachment to the bone endothelium. Several promising candidate receptors were found in his mouse system, which will be tested in the future using peptides or peptide derivatives for inhibition of cancer cell spread.
Dr. Hillary Nelson at the University of California, Berkeley completed a one-year IDEA grant entitled Structure of Est-1: a Potential Drug Target for Breast Cancer to develop information on a protein factor that permits the unlimited growth of breast cancer cells. The telomerase enzyme complex repairs the ends of chromosomes after cell division and is present in the majority of breast cancers at a very early stage. A telomerase accessory protein, est-1, regulates telomerase assembly and activity, and could be a target to inhibit cancer cell growth. Dr. Nelson made initial efforts to produce and purify large amounts of est-1 for her studies.
In his study entitled Biology of Telomere Length Conservation in Breast Cancer, Dr. Darryl Shibata at the University of Southern California investigated the activity of the telomerase enzyme with respect to breast cancer progression. Telomerase has gained recent attention in cancer as a target to limit cancer cell immortality in order to arrest tumor growth. In his one-year IDEA project, Dr. Shibata found that telomerase activity is heterogeneous in different areas and cells of a tumor mass, and telomerase activity appears early in breast cancer progression. This project combined both a molecular approach with an emphasis on information derived from clinical material, a valuable multidisciplinary perspective.
Dr. Marian Waterman at the University of California, Irvine in her one-year IDEA project, Proper Relocation of a Tumor Suppressor in Breast Cancer, investigated mechanisms used by breast cancer cells to localize regulatory proteins with the cell. She was particularly interested in the BRCA 1 protein, which when mutated is associated with a high risk for breast and ovarian cancer. Dr. Waterman determined two sites on BRCA 1 that interact with certain cellular localizing proteins. This study also supported the concept that BRCA 1 is a likely tumor suppressor, and these transport proteins target BRCA 1 to the cell nucleus. Further work on cellular transport proteins will be important to reveal how key gene regulatory and tumor suppressor genes function in breast cancer cells.
Research in Progress
Outbreak — how cancer spreads: angiogenesis, invasion, and metastasis
Physical tumor growth is accompanied by recruitment of blood vessels (angiogenesis) as a precursor to the spread of tumor cells to distant sites in the body (metastasis). The specific breast cancer cell surface receptors necessary for spread in the body are being studied by Dr. Jeffrey Smith of The Burnham Institute. His laboratory has found that certain ‘integrin’ receptors on breast cancer cells are required for metastasis, and is now investigating possible regulatory proteins that control the binding function of tumor integrins. Dr. Alex Strongin at the La Jolla Institute of Experimental Medicine is looking at ways to inhibit metastasis by examining MMP-2 collagenase activation. They found that active collagenase MMP-2 modulates the invasion of tumor cells not only by degrading tissue proteins, but also by masking certain specific cell surface receptors, integrins, by which tumor cells recognize these tissue proteins. They identified the region of MMP-2 that directly interacts with integrins.
Some proteins can serve as predictors of breast cancer development and progression. Understanding how known markers function in normal and tumor cells and identifying new markers can provide us with ideas regarding what aspects of cell growth and development should be targeted for therapy. Dr. Pierre Desprez at the California Pacific Medical Center looks at the role of Id-1 in breast cancer metastasis. He found that the presence of Id-1 is associated with the invasive phenotype and can cause non-invasive cells to become invasive when Id-1 protein is present. Dr. Daisy De Leon of Loma Linda University examines the role of IGF-II and cathepsin D in tumor metastasis. She asked two questions; do high levels of IGF-II and cathepsin D increase tumor growth and metastasis, and do human breast tumors produce high levels of IGF-II and specific forms of cathepsin D? She found that a distinct form of Cathepsin D is present in high amounts in breast cancer by examining normal and tumor tissues from the same patient.
Too much cell growth: defective messages and internal signaling
Estrogen exposure is strongly associated with the development of breast cancer. Dr. Shiuan Chen at the Beckman Research Institute of the City of Hope has studied how estrogen is produced in fat tissues. He found that cancer-free areas of the breast produce estrogen in lesser amounts than areas of the breast with cancer. He also found that the regulation of estrogen production was under the control of glucocorticoid hormones in cancer-free areas, whereas a protein called cAMP regulated estrogen production in areas with cancer. This study provides evidence that there may be a fundamental difference in how breast tumors are provided with estrogen for their growth when compared with normal breast cells. We know that, in order to function, estrogen must bind to its receptor (ER). Dr. Michael Stallcup at the University of Southern California has found novel protein fragments (GRIP2 and GRIP3) that could be used to block the association of the estrogen receptor with its growth promoting coactivators.
Tamoxifen therapy is commonly used in patients with estrogen responsive tumors. Dr. Myles Cabot at the John Wayne Institute for Cancer Treatment has 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 evidence that tamoxifen can reverse chemotherapeutic resistance.
Growth of breast tumors is influenced by cell surface receptors that present attractive targets for therapeutic intervention. Basic research on these receptors is still a pre-requisite for developing novel approaches in the future. The Erb2/Her/Neu growth receptors activate genes through intracellular pathways of signal transduction. Dr. Daniel Donoghue at the University of California, San Diego is studying how the critical Neu/Her2 receptor associates as a dimer, which is an active form. He has found protein sequences in the transmembrane domain of Neu/Her2 that are necessary for dimerization. In a separate project, Dr. Gordon Gill at the University of California, San Diego examines the role of sorting nexins, which can control cell growth by degrading receptors. They have found a new sorting nexin (SNX2) that has a different specificity than the SNX1. SNX1 specifically degrades EGF receptors. This means that different sorting nexins can be targeted to different receptors. There is a family of sorting nexins that have similar functions but affect different growth controlling pathways.
In the normal course of events, cells perform their function and then die at the appropriate time a process called programmed cell death or apoptosis. One important way that tumor cells and normal cells differ is that tumor cells do not die off as rapidly as they should. Research examining why tumor cells are deficient in this ability should eventually lead to discovering ways to cause tumors to reacquire this ability. Dr. Michael Karin at the University of California, San Diego and his Postdoctoral Fellow, Dr. Zheng-gang Liu, are studying the Mitogen Activated Protein Kinase (MAPK) signaling pathways in breast cancer cells. They report that "activation" of the NF-kB transcription factor via tumor necrosis factor (TNF) and MAPKs allows a breast cancer cell to evade programmed cell death. Dr. Jamil Momand at the City of Hope National Medical Center is investigating the loss of p53 function in breast cancer and the ability of a natural inhibitory protein MDM2 to prevent p53 from entering the cell's nucleus to perform its normal functions. Dr. John Reed at The Burnham Institute is investigating how suicide receptors Tumor Necrosis Factor Receptor-Associated Factor (TRAF) proteins transfer suicide signals from TNF receptors into breast cancer cells. He has found that breast cancers downregulate their levels of TRAF-4 suggesting an important change in these tumors that may facilitate their escape from immune surveillance.
Mistakes on the master blueprint: molecular genetics and gene regulation
Dr. Utha Hellmann-Blumberg at the University of California, Davis has found that tamoxifen can be metabolized into a product (4-hydroxy, N-desmethyltamoxifen) that binds to DNA, which is the first step in chemical carcinogenesis, and that this product is detectable in patients taking tamoxifen. Additionally, breast tumors develop resistance to tamoxifen over time and some even become responsive to it. Understanding how tamoxifen interacts with estrogen to exert its effect is crucial for determining the parameters for safe and effective use of tamoxifen, as well as the development of more effective antiestrogens. This line of investigation requires that we also develop an understanding of how estrogen works. Dr. Robert Oshima of The Burnham Institute is investigating a transcription factor Ets2, thought to be capable of switching on or off critical genes necessary for breast cancer progression. He is using transgenic mice as a way to address fundamental questions not approachable using cell lines and tumor samples. On a more global level, breast cancer cells may organize their chromosomes differently than normal cells. Dr. Helene Baribault of The Burnham Institute is using transgenic mice having defective apoptosis regulatory protein Bax, to determine why it appears to be critical for certain therapies for breast cancer in women. Dr. John Reed of The Burnham Institute is devising strategies for enhancing the function of Bax to treat breast cancer.
Searching the unknown: novel breast cancer genes
Breast cancer cells express and regulate their genetic information differently from normal cells. Dr. Michael Press at the University of Southern California is investigating the genes that cause cancer cells to accumulate abnormally large amounts of DNA by finding human gene equivalents to rum1 (the yeast gene that regulates the DNA accumulation). They have found two genes in human cells that qualify as human cousins to rum1. Work is now progressing to determine how these genes function in the cell cycle. Dr. Gregory Shackleford at the Childrens Hospital Los Angeles is identifying genes that are turned on during tumor formation in a mouse model with a predisposition to breast cancer. They are developing a transgenic mouse model that carries two breast cancer genes that are known to cooperate with each other to form tumors (FGF and Wnt) and using the transgenic mice to identify novel genes that cooperate with FGF and Wnt to form tumors more efficiently. So far they have found several genes for factors that cooperate with Wnt in mice and are testing human breast cancers for the activation of these genes. Dr. Terumi Kohwi-Shigematsu at the Lawrence Berkeley National Laboratory is studying proteins from breast cancer cells that organize their chromosomal structure. Her laboratory has found specific matrix organizing region binding proteins specific to breast cancer that could be therapeutic targets.
Unraveling the path to breast cancer: tumor progression
Dr. Valerie Weaver at the Lawrence Berkeley National Laboratory is investigating the role of Vitamin D to restore proper communication of breast cancer cells with their microenvironment, and she finds that a specific integrin receptor is necessary for these tumor functions. She can revert tumor cell functions to normal by using specific antibodies to these integrins.
Recently Initiated Research
In 1997, 22 new grants were funded to further understanding of how breast cancer develops, including identifying and understanding the action of genes involved in cell growth control, determining actions of hormones and hormone-blocking drugs, exploring why breast cancer spreads to other parts of the body, looking at effects of vitamins on breast cancer cells, An additional five grants were funded to explore the basic biology of the human breast, including effects of pesticides, repair of genetic damage, and roles of vitamins and hormones on normal breast cells.
