Pathogenesis: Understanding the Disease

Although cancer is often described simply as a genetic disease, there are many competing theories to explain the gene alterations and mutations that initiate cancer and those that promote disease progression. W. Wayt Gibbs, writing in the July 2003 issue of Scientific American, summarized this cancer confusion as, “…it is more useful to think of cancer as the consequence of a chaotic process, a combination of Murphy’s Law and Darwin’s Law: anything that can go wrong will, and in a competitive environment, the best adapted survive and prosper.” In a more scientifically detailed fashion, Dr. William Hahn at Dana-Farber Cancer Institute in Boston and Dr. Robert Weinberg at MIT have pointed to six key cellular events that are “hallmarks of cancer” (recently reviewed in New England Journal of Medicine 348 (2003):674). Taken together these cellular events attempt to account for the sporadic nature of cancer; the biological and pathological heterogeneity seen in patients; immortality of cancer cells; numerous gene and chromosomal alterations; uncontrolled growth, motility, and metastasis events; and resistance to therapy.

Since no single approach can successfully explain cancer, researchers are employing a variety of technologies and methods. These range from cell and animal models; complex genomic and proteomic techniques to identify and relate multiple genetic changes in various forms of the disease; and the application to cancer of new discoveries in basic cell biology, DNA repair, cell cycle, growth signaling, and gene regulation processes. Still, in breast cancer the response to hormones, especially estrogen, remains a key underlying theme. Researchers now think that estrogen may operate in ways outside the classical estrogen response. New thinking is also emerging for the growth factor receptors, epidermal growth factor receptors (EGFR), and the Her oncogenes. There is much interest in cross-talk between the hormone response and growth factor signaling pathways, previously thought to be distinct. Finally, the inherited breast cancer risk genes, BRCA1 and BRCA2, are being studied in more advanced ways to better explain how DNA defects, repair processes, and cell growth/death pathways are interrelated and become defective in cancer.

Research Conclusions

Under the Pathogenesis priority topic 19 grants were completed in 2003.

A New Model for Inflammatory Breast Cancer.
Anti-E-Cadherin Apoptosis of Inflammatory Breast Carcinoma.
Inflammatory breast cancer is a relatively rare, fast-growing form of breast cancer usually not detected by mammograms or ultrasound. Unlike other types of breast cancer, inflammatory breast cancer spreads locally in the breast via the lymphatic system just below the skin. To better understand how the inflammatory breast cancer cells enter the blood and lymphatic vessels, the CBCRP has funded Sanford Barsky, M.D., and a postdoctoral fellow in his laboratory, Mary Alpaugh, Ph.D., from the University of California, Los Angeles. Together they developed a method for getting an inflammatory breast tumor to grow in a mouse’s lymph and blood vessels the same way it does in humans. They have used this transplantable tumor, called Mary-X, to look at how the protein E-cadherin (which is produced by normal breast cells) will allow the cells to attach in layers to other cells, functions in the inflammatory breast tumor, and how stopping this attachment affects these cells. They were also able to learn more about what makes inflammatory breast cancer cells resistant to chemotherapy and why these cells easily spread to other parts of the body. Future research with the transplantable tumor, Mary-X, may lead to new treatment options for inflammatory breast cancer. It also will allow researchers to study how all forms of breast cancer are able to spread through the blood and lymph system to other parts of the body. Research findings supported by CBCRP funding to Drs. Barsky and Alpaugh appeared in many publications, including Cancer Research 61 (2002):5231-41, Oncogene 21 (2002):3631-43, Human Gene Therapy 13 (2002):1245-58, and the American Journal of Pathology 161 (2002):619-28. Dr. Barsky has described his concept of tumor growth as the “ship in a bottle” to suggest that tumors (ship) induce the surrounding stroma and blood vessels to create the vascular “bottle” around themselves.

Role of MMPs in Breast Tumor Initiation and Aggressiveness.
Metalloproteinases (MMPs) are enzymes that normal cells secrete to allow cell movement, tissue remodeling, and healing processes. Breast cancer cells produce more MMPs than normal and this allows tumors to become invasive. Jimmie Fata, Ph.D., of the Lawrence Berkeley National Laboratory, investigated how high levels of a specific MMP, called MMP-3, cause breast cancer in mice. He explored whether MMP-3 is able to split the endothelial protein E-cadherin, which attaches cells to one another. This splitting could lead normal breast epithelial cells (the type of cells where most breast cancer begins) to develop an unusual characteristic called epithelial to mesenchymal transition (EMT). EMT allows cells to spread to other body parts and is associated with aggressive breast cancers. Dr. Fata created cells with EMT characteristics, and then inhibited the action of MMPs in these cells. He also developed cells with mutant forms of a cell-cell adhesion protein, called E-cadherin.
Dr. Fata found forms of E-cadherin that were resistant to MMP splitting. Dr. Fata, his mentor Dr. Mina Bissell, and Dr. Zena Werb from the University of California, San Francisco, reviewed this topic in Breast Cancer Research 6:1-11.

Metastasis Suppressor Genes for Breast Cancer.
Are there cancer metastasis genes that have not yet been discovered, and do genes exist that work as tumor suppressors to effectively block cells from spreading? These two questions were addressed by Stanley Cohen, M.D., at Stanford University. Dr. Cohen and his laboratory are pioneers in gene cloning technology, and he used CBCRP funding to turn his attention to breast cancer. Using a technique called Random Homologous Knock Out (RHKO), Dr. Cohen has been able to isolate cancer cells with defective metastasis suppressor genes, which allow the cancer cells to spread. While doing this research Dr. Cohen found an oncogene, a gene that causes normal cells to change into cancerous cells, that is clearly associated with metastasis. Dr. Cohen also has begun working with a biotech company to develop ways to externally detect very small tumors in mice being used in cancer research. This external detection method would allow researchers to determine whether a tumor has spread without killing or dissecting the mice, thereby allowing researchers to obtain more comprehensive analyses from their studies. Dr. Cohen’s work with metastasis suppressor genes (MSGs), the oncogene he found, and the external detection system have the potential to contribute to the identification of new approaches to slowing or stopping breast cancer metastasis. Use of the technology to detect novel tumor suppressor genes was published by Dr. Cohen and his CBCRP-supported postdoctoral fellow, Dr. Quan Lu, in the Proceedings National Academy Sciences USA 100 (2003):7626-31.

Novel Enzymes Associated with Breast Cancer Angiogenesis.
Lasp-1 Signaling in Breast Carcinoma Cell Invasion/Migration.
Hox Transcriptional Regulation of Angiogenesis.
Three CBCRP grants were funded to study novel genes and processes in relationship to breast cancer angiogenesis. Steven Rosen, Ph.D., of the University of California, San Francisco, studied two novel enzymes, Sulf-1 and Sulf-2. First, they cloned cDNAs and sequenced Sulf-1 and Sulf-2 proteins in both mice and humans. This allowed them to look closely at how the enzymes function and to confirm that they are involved in the angiogenesis process. Then, Dr. Rosen’s lab demonstrated that there are large amounts of Sulf-2 in mice with breast cancer and in human breast cancer. These findings led Dr. Rosen to conclude that it is possible that the Sulfs play a role in promoting the creation of new blood vessels in tumors. Results from this grant were published in the Journal of Biological Chemistry 277 (2002):49175–185.

The Lasp-1 gene is associated with breast cancer invasion in humans. Clinical studies have found that the Lasp-1 gene is turned on in 8–12 percent of breast cancer cells. Yi Hsing Lin, Ph.D., at the Scripps Research Institute, La Jolla, had already shown that overproduction of the protein produced by Lasp-1 was necessary for cells to move to other body parts. The next step was to determine which part of the Lasp-1 protein was responsible for the spread of breast cancer. Dr. Lin’s team found that two types of proteins—growth factor proteins and extracellular matrix proteins—played a role in getting Lasp-1 to get cells to move to other areas.

Audri Charboneau, Ph.D., at the University of California, San Francisco, investigated two developmental genes, Hox D3 and Hox D10, which control the vascular endothelial cells and their role in angiogenesis. Her research explored whether sustaining high levels of the protein produced by the Hox D10 gene, which keeps the endothelial cells quiet, or stopping the protein produced by the Hox D3 gene, which makes these cells active, affects the development of new blood vessels. She also studied possible ways of preventing the migration of the endothelial cells, which would keep the cancer from spreading. By removing Hox D3 or introducing Hox D10 into endothelial cells, they were able to block new blood vessel development.

Molecular Characterization of ErbB2 Positive Breast Cancers.
The Role of SGK in Breast Cancer Cell Proliferation.
The ErbB2 (Her-2/neu) protein is present at high levels in 20–30 percent of all breast cancers, but is more common in more aggressive cancers, especially breast cancers that do not have estrogen receptors. Two completed CBCRP grants studied genes that are associated with ErbB2 (Her-2/neu) and could provide clues on its role in breast cancer. Richard Neve, Ph.D., of the Buck Institute for Age Research, Novato, is trying to find a better way to classify ErbB2-positive breast cancers into distinct molecular subtypes, so that effective therapies could be developed for each subtype. He is studying a generegulatory transcription factor protein, called ESX that is found in cancer cells that are high in ErbB2. Dr. Neve’s team analyzed ErbB2 and ESX levels in 45 primary breast cancer samples and explored how the proteins interact with one another. They found that ESX caused significant changes in the epithelial cells in the breast where most cancer begins, and they identified a group of genes controlled by ESX and ErbB2. In addition, they found a group of proteases (a type of enzyme) that have the ability to help cancer cells spread and that respond in high levels to ESX. Dr. Neve has moved to the University of California, San Francisco, and he is pursuing work on ESX and ErbB2 to confirm theses findings. Results from his project were published in Oncogene 21:3934-8.

Masaaki Hayashi, M.D., Ph.D., at The Scripps Research Institute, La Jolla, explored how ErbB2 gets cancer cells to grow. He investigated two proteins that help ErbB2 send signals to cancer cells, glucocorticoid-inducible kinase (SGK), which is necessary for breast cancer cells to grow, and the protein, Big MAP Kinase (BMK1), which regulates SGK. He found that there was a specific part of the BMK1 protein that is activated by SGK in breast cancer cells, and he identified 12 other proteins that interact with SGK. He also found that when SGK is shut off in breast cancer cells, those cells stop reproducing, demonstrating that SGK is necessary for cancer growth. Dr. Hayashi also developed a special mouse model that can be used for additional research on the proteins BMK1 and SGK.

Genetic Analysis of ErbB Signaling in C. Elegans.
In mammals, the ErbB family of genes regulates the development of breast tissue, and corresponding (homologous) genes exist in lower organisms. Since fundamental cell processes (such as the cell cycle, DNA repair, and development) are conserved in evolution, researchers find it instructive to step back to organisms with a simpler biology to study genes and proteins that are important in human disease. Nadeem Moghal, Ph.D., at the California Institute of Technology, Pasadena, used a nematode worm, called C. elegans, as a model system to identify and analyze which genes interact with the ErbB genes and the proteins they produce. Dr. Moghal’s mentor, Dr. Paul Sternberg, uses this approach to study behavior, cell spatial patterns, and organ formation. After injecting the worms with a chemical that causes mutations, Dr. Moghal studied what happened to the worms’ pseudo-ErbB gene, called let-23. This allowed him to identify a gene (TRAP230) whose mutation might play a role in the development of breast cancer and to define new mutations that might make ErbB genes too active. He also demonstrated that abnormal gene activity in other tissue, like muscle, could affect how ErbB responds in the breast’s epithelial cells. Dr. Moghal intends to conduct future research on ErbB proteins and the TRAP230 gene. His work was published in Oncogene 22 (2002):5471-80, Development 130 (2003):4553-66, Experimental Cell Research 284 (2003):150-9, and Development 130 (2003):57-69. Dr. Moghal is continuing his research in oncology and use of C. elegans at the University of Utah, Huntsman Cancer Institute.

Molecular Study of BAG Domains: A New Motif in Breast Cancer.
In cell signaling processes, proteins interact by binding to each other in structurally compatible regions. Essentially, they assemble into multi-protein, functional complexes like pieces of a puzzle. Structural biologists dissect these protein-protein binding events by such techniques as nuclear magnetic resonance (NMR) imaging and crystallography. This approach is ideal to study apoptosis, because the processes of programmed cell death involve a number of key protein interactions, and identifying the sub-domains involved could lead to a rapid development of cancer therapeutics. Klara Briknarova, Ph.D., at The Burnham Institute in La Jolla, investigated proteins from the BAG protein family. The initially-described member of this family, BAG1, is present in elevated levels in many breast cancers, promotes tumor growth and spread to other body parts, and makes tumors resistant to anticancer drugs such as tamoxifen. All of the BAG proteins have their activity directed by the BAG domain. Working with her mentor, Dr. Kathryn Ely, Dr. Briknarova was able to determine the molecular structure of the BAG domain and to closely study how the BAG1 and BAG4 proteins operate. Results from this project were published in the Journal of Biological Chemistry 277 (2002):31172-8.

Overcoming Drug Resistance in Breast Cancer.
Breast cancer cells can sometimes avoid being killed by chemotherapy, and researchers are studying the underlying biological reasons for this resistance. It is believed that most chemotherapy drugs work by triggering cell death, called apoptosis, and the adhesion status of the cell may influence this process. Exploring this hypothesis, Kristiina Vuori, M.D., Ph.D., from The Burnham Institute, La Jolla, found that there are certain molecules on the cell surface, known as integrins, that not only keep cancer cells from getting the signal that they should die, but may even make cancer cells resistant to chemotherapy. Dr. Vuori found that she was able to use anti-integrin antibodies to stop the integrins from blocking the message telling the cell to die. She also learned more about the integrins’ ability to turn on an enzyme called PI3- kinase, which helps send the “do-not-die” message. Dr. Vuori’s continued research in this area could lead to new ways of increasing the benefits of chemotherapy. Results from this research were published in Oncogene 20 (2001):4995-5004.

The Role of BRCA1 in Nucleotide Excision DNA Repair.
Nucleotide excision repair (NER), a type of DNA repair pathway, corrects DNA damaged from many environmental toxins, including cigarette smoke and ultraviolet radiation. The tumor suppressor gene, p53, and the breast cancer hereditary gene, BRCA1, are both involved in NER, and NER is subdivided into two pathways—global genomic repair (GGR)—which targets and removes lesions from the whole genome, and transcription-coupled repair (TCR), which preferentially removes lesions from the transcribed strand of expressed genes.

Anne-Renee Hartman, M.D., at Stanford University, addressed the question of how BRCA1 affects NER and whether this effect is independent of p53. She found that BRCA1 plays a role in maintaining the NER pathway in the cell. This effect is very significant when the p53 tumor suppressor gene is not functioning in the cell, which occurs in more than 50 percent of human cancers and more than 80 percent of BRCA1-associated breast cancers. Dr. Hartman demonstrated that BRCA1 affects NER through transcriptional regulation of NER genes. These findings could support the development of clinical trials for women with BRCA1 mutations using specific chemotherapy drugs, like cisplatin, that specifically target the DNA repair process. Results of the study were published in Nature Genetics 32 (2002):180-4 and the Journal of Molecular Medicine 81 (2003):700-7.

DNA Packaging Defects in Breast Cancer.
If unraveled, the chromosomal DNA from a single cell would stretch about three meters. Obviously, there are structural solutions that cells use to package this DNA into a nucleus that is only a few microns (10-6 M) in diameter. Terumi Kohwi-Shigematsu, Ph.D., from the Lawrence Berkeley National Laboratory, studied the differences in the DNA structure and packaging between breast cancer cells and normal cells. She identified a protein called poly (ADP-ribose) polymerase (PARP) that binds to a specific stretch of DNA, called the matrix attachment regions (MARs). Dr. Kohwi-Shigematsu’s research demonstrated that the PARP protein, which plays a role in DNA repair, is found in high levels in breast cancer cells but at very low levels in normal cells. She also found that when PARP is removed from aggressive human breast cancer cells that are being studied in the lab, the breast cancer cells change back into the type of cells that are less likely to invade other tissues. To investigate this further, Dr. Kohwi-Shigematsu used microarray technology —a technique that allowed her to simultaneously check 20,000 genes—to look for the specific genes that were turned on in both the aggressive breast cancer cells and those that had had PARP removed. This revealed several genes that play a role in cancer development. In addition, Dr. Kohwi-Shigematsu studied mice that had been genetically engineered to spontaneously form breast cancer but to not produce PARP. Preliminary data from this research suggests that mice are less likely to grow tumors when PARP is not present. Taken as a whole, this research suggests that PARP helps cancers grow and that targeting PARP may be beneficial for cancer treatment. Dr. Kohwi-Shigematsu received a new CBCRP grant in 2002 to study this further. Publications based on this research appeared in the Journal of Cellular Biochemistry 35 (2001):36-45 and Critical Review Eukaryotic Gene Expression 10 (2000):63-72.

TGF-b3 and Small GTPases in Invasive Breast Cancer.
Vesa Kaartinen, Ph.D., of the Children’s Hospital Los Angeles, investigated proteins involved in the epithelial-to-mesenchymal transdifferentiation (EMT) process, and focused on two GTPases called Rac and Rho. GTPases are proteins that can control a chain of chemical reactions within a cell. Some GTPases tell a cell to divide and grow; others tell a cell to move from one location to another. Dr. Kaartinen found that the protein TGF-?3 changes the locations and amounts of two adhesion molecules (integrins) in mouse mammary epithelial cells. The team also found that Rac3, a protein involved in a chain of chemical reactions within these cells, plays a role in the growth of mammary epithelial cells and in the formation of cells that are in transition from normal to cancerous. Dr. Kaartinen intends to continue to study the role of TGF-?3 and GTPases in invasive breast cancer. Results of this study were published in International Journal of Molecular Medicine 9 (2002):563-70 and the Journal of Biological Chemistry 277 (2002):8321-8.

Immortalization of Human Mammary Epithelial Cells by ZNF217.
Amplification of the ZNF217 gene, meaning that more than two copies are present, has been found in many different types of tumors, including some 40 percent of human breast cancer cell lines. Paul Yaswen, Ph.D., at the Lawrence Berkeley National Laboratory, found that higher than normal levels of ZNF217 not only helps keep cells alive but also may make the cells resistant to chemotherapy and radiation treatments. Dr. Yaswen and collaborators will continue to study ZNF217 to determine if there is a relationship between how active a woman’s ZNF217 gene is and her risk for breast cancer or her prognosis after she is diagnosed with breast cancer. If ZNF217 is found to be involved very early on when breast cancer develops, then this research could lead to the development of new drugs that could prevent the disease from occurring. Results from this research were published in the International Journal of Biochemistry and Cell Biology 34:1382-94 and Cancer Letters 194 (2003):199-208.

Rodent Model for Human Ductal Carcinoma in Situ.
Mammography has greatly increased the detection of two types of pre-cancers:
ductal carcinoma in situ (DCIS) and lobular carcinoma in situ (LCIS). Although almost all breast cancer begins as DCIS or LCIS, not all DCIS or LCIS will become breast cancer. Currently, however, there is no way to tell which pre-cancers will progress and become cancers. To help unravel some of the questions about DCIS and LCIS, Satyabrata Nandi, Ph.D., of the University of California, Berkeley, developed a method to induce a large variety of DCIS and LCIS in rats. Dr. Nandi then demonstrated that it is possible to use a cancer-causing chemical to make the DCIS and LCIS become breast cancer. This research will allow Dr. Nandi to conduct further studies on DCIS and LCIS and how hormones influence their growth. These studies could ultimately lead to better ways of determining which DCIS and LCIS will progress to cancer and how DCIS and LCIS should be treated.

Role of PTEN/Akt Pathway in Invasion in Human Breast Cancer.
Ductal carcinoma in situ (DCIS) is considered a pre-cancer and not true cancer because the altered cells are confined to the breast duct. It is known that about 25–30 percent of DCIS lesions will eventually progress to become invasive cancer, but it is not known how to predict which cases have the potential to become invasive. Shikha Bose, M.D., at Cedars-Sinai Medical Center in Los Angeles, believes that there are underlying genetic differences in DCIS that might be used to predict which DCIS cases would progress to invasive cancer. This research has focused on PTEN, a recently identified tumor suppressor gene that is located in a part of chromosome 10 and that is frequently lost in invasive breast cancer. When PTEN becomes lost or altered because of changes to chromosome 10, it creates the possibility for invasive breast cancer to occur. The team’s research focused on surveying the global genetic changes that link PTEN with breast cancer invasion and progression. By cDNA array comparisons they will be able to catalog and compare genetic changes in women with DCIS to those with invasive breast cancer. The goal is to validate genetic markers that physicians could use when making treatment decisions. These genetic markers might also provide insight into which proteins and genes could be investigated for new drug development. Dr. Bose is continuing this research with additional CBCRP funding that began in 2003.

Studies of Telomere Capping Dysfunction in Breast Cancer.
Protective caps, called telomeres, keep chromosomes in working order. David Gilley, Ph.D., at the Lawrence Berkeley National Laboratory, tested the hypothesis that breast cancer begins when problems with the telomeres lead the chromosomes to become uncapped. This uncapping could allow chromosomes that should be separate to fuse together, leading to genetic mistakes that can result in the development of breast cancer. His team found that an important telomere protein, called TRF2, is found at higher levels in breast cancer. This suggests that TRF2 may play a role in the development of breast cancer. Dr. Gilley intends to continue to study TRF2 to learn more about its role in breast cancer. This work could lead to a better understanding of how breast cancer begins. Dr. Gilley resigned his New Investigator CBCRP award after one year to accept a faculty position at Indiana University.

Role of p53 in Irradiated Stroma and Mammary Carcinogenesis.
Ionizing radiation, such as x-rays, can cause changes in breast cells that lead them to become cancerous. Most studies concentrate on the changes in epithelial cells, where breast cancer begins. Mary Helen Barcellos-Hoff, Ph.D., at the Lawrence Berkeley National Laboratory, pursued the hypothesis that radiation may cause changes in the stromal cells that are part of the framework that supports epithelial cells. These changes, in turn, may create an environment that makes the epithelial cells more likely to become cancerous. Working with Dr. Joseph Jerry at the University of Massachusetts, Amherst, Dr. Barcellos-Hoff’s team used mouse mammary epithelial cells that lack a gene, p53, which normally suppresses tumors, to determine the exact radiation exposure that would lead to cancer development. They found that a radiation dose of 4 Gy did not change the length of time it took tumors to develop or how often they developed, but that it did change the characteristics of the tumors that did develop. They also made the surprising observation that doses of radiation less than 4 Gy actually reduced the number of tumors that developed. This research on how radiation alters normal mechanisms that stromal cells use to keep epithelial cells from turning cancerous may provide new strategies for enhancing these mechanisms to prevent or reverse cancer. She has published her work in the Journal of Mammary Gland Biology and Neoplasia 2001 Apr; 6(2):213-21.

Research in Progress

A number of ongoing CBCRP grants in the topic of Pathogenesis reported substantial progress in 2003.

Profiling Enzyme Activities in Models of Human Breast Cancer.
Benjamin Cravatt, Ph.D., from The Scripps Research Institute, La Jolla, has developed a novel technique called activity-based protein profiling (ABPP) that is able to detect, measure, and purify active breast cancer-associated enzymes. This proteomic-based approach is significant, because the traditional, biochemical approach to investigate proteases could not reliably detect active forms from those present as precursors (zymogens) or bound to inhibitors. Five papers on Dr. Cravatt’s research were published, including articles in the Journal of the American Chemical Society 125 (2003):4686-4687 and Proceedings of the National Academy of Sciences USA 99 (2002):10335- 10340.

The Detailed Structure of a Model Breast Cancer Genome.
Locating Novel Breast Cancer Genes Using DNA Microarrays.
Two ongoing CBCRP-funded grants are aiming to apply novel technologies to map and catalog the chromosomal changes in breast cancer cells and tumor samples. Colin Collins, Ph.D., at the University of California, San Francisco, is using a new technique called End Sequence Profiling that has the potential to identify all the genetic differences between breast cancer and normal cells. End Sequence Profiling uses some of the same methods that were used to map the human genome. Dr. Collins begins by creating a bacterial artificial chromosome (BAC) library for the tumor being studied. He then compares the BAC with a reference library of chromosomes, which allows him to quickly see if there are extra genes or missing ones. Results from Dr. Collin’s CBCRP project were published in the Proceedings of the National Academy of Sciences USA 100 (2003):7696-7701 and Bioinformatics 1 (2003):1-12.

Jonathon Pollack, M.D., Ph.D., at Stanford University School of Medicine, is using the more established method of DNA microarrays (gene-chips) that can look simultaneously at more than 26,000 genes from human tumor samples to find extra and missing genes. In collaboration with CBCRP-funded investigators, such as Dr. Stefanie Jeffrey at Stanford, Dr. Pollack’s laboratory can directly compare expression data (i.e., messenger RNA assays) with chromosomal data to determine whether genetic alterations associated with gene deletion or duplication result in a corresponding change in the gene transcription. This research could lead to new genetic tools that will help oncologists assess how aggressive a cancer is and improve treatment options. Genetic approaches to classifying breast cancers were reviewed by Drs. Pollack and Jeffrey in Breast Cancer Research 5 (2003):320-8.

Cyclin E Affects Growth Arrest in Breast Cancer Cells.
Caspase-mediated Apoptosis Mechanisms in Breast Cancer Cells.
Regulation of the Rad1 Checkpoint Complex in Breast Cancer.
Three CBCRP dissertation grants to support graduate students achieved breakthrough findings in 2003. Navdeep Dhillon, at the University of California, Davis, is investigating the cell cycle regulatory protein, called cyclin E, and the role it plays in breast cancer and programmed cell death (apoptosis). Dhillon and colleagues found that cells that contain high levels of cyclin E do not respond as well to tamoxifen. They also found that elevated levels of cyclin E result in a severe decrease of a protein that prevents cell death. This has led Ms. Dhillon and her mentor, Dr. Maria Mudryj, to propose a novel function for the cyclin E protein as an “effector” for cell death. Results from their research were published in Genes and Immunity 4 (2003):336-342.

Kelly Boatright, from the Burnham Institute, La Jolla, has developed a technique for studying the key apoptosis cell death enzymes, called caspases. Her methods allow the comparison of caspase function in normal cells versus cancerous breast cells. She is also investigating whether cancer cells are more likely to respond to an anti-tumor agent called TRAIL (TNF-related apoptosisinducing ligand) when it is combined with the chemotherapy drug doxorubicin (Adriamycin). This work could lead to new ways of inducing cancer cells to respond better to cancer treatments. Results from this research in the laboratory of Dr. Guy Salvesen were published in Molecular and Cellular Biology 11 (2003):529-541 and the Journal of Biological Chemistry 278 (2002):10458-10464.

Cells are constantly exposed to agents that can damage their DNA. Checkpoint proteins in cells sense and respond to DNA damage and slow cell growth until the damage is repaired. The risk for cancer increases when these checkpoint proteins fail to work. Patrick Lupardus, at Stanford University School of Medicine, is investigating how two checkpoint proteins, ATR and Rad1, interact to start the DNA repair process. Greater understanding of these checkpoint proteins and how they work may provide new opportunities to prevent or treat breast cancer in ways that—unlike chemotherapy—do not harm normal healthy cells. Results from this research performed in the laboratory of CBCRP-funded investigator Dr. Karlene Cimprich were published in Genes and Development 16 (2002):2327-2332.

Structure and Function of the Bax Apoptosis Regulator.
The Bcl-2 family includes some proteins that can activate the process of cell death (apoptosis) and others that can stop it in normal cells and breast cancer cells. Francesca Marassi, Ph.D., at The Burnham Institute, La Jolla, is investigating two members of the Bcl-2 family, Bc1xL and Bax. Bc1xL keeps cells from dying while Bax promotes cell death. Dr. Marassi’s hypothesis is that these proteins play a role in controlling whether breast cancer cells die. The information gained from this research will provide new information about the normal breast and breast cancer, and may be useful for developing new approaches to breast cancer treatment. Results from this research were published in five journals, including Biochemica et Biophysica Acta 1645 (2003):15-21, Journal of Magnetic Resonance Imaging 161 (2003):64-69, and Protein Science 12 (2003):403-411.

Genes That Modulate Dioxin-Induced Breast Cancer.
Dioxins are widespread environmental toxins known to cause cancer. They are accumulating in foods, including breast milk. Several studies suggest dioxin may be responsible for some breast cancer cases. Quan Lu, Ph.D., of Stanford University, searched for genes that either promote or suppress breast cancer initiated by dioxin. The research team used two techniques. The first, RHKO, has been used to discover genes that inhibit tumor growth. The second, microarrays, is a technology that allows a researcher to study thousands of genes simultaneously. These techniques allowed Dr. Lu to identify several previously unknown genes that are involved in cancers caused by dioxins. Use of the technology to detect novel tumor suppressor genes was published by Dr. Lu and his mentor Dr. Stanley Cohen in the Proceedings National Academy Sciences USA 100 (2003):7626-31.

BRCA1-Dependent Ubiquitin Ligase Activity in Breast Cancer.
The BRCA1 gene is gaining an established role in DNA repair, but it also functions in the regulation of cellular protein turnover. BRCA1 has “ubiquitin ligase” activity, and Yan Xia, Ph.D., at the Salk Institute for Biological Studies, La Jolla, is studying this function of this BRCA1-regulated process. Results from this research were published in the Journal of Biological Chemistry 278 (2003):5255-63.

Cell-Killing Effect of Orphan Receptor TR3 in Breast Cancer.
Vitamin A compounds, called retinoids, are being studied for their ability to prevent or treat cancer. Research has shown that one of these compounds, called AHPN, can cause breast cancer cells to die, and that a protein called TR3 plays an important role in this process. Nathalie Bruey-Sedano, Ph.D., at The Burnham Institute, La Jolla, is investigating what chemical reactions within cells are necessary for TR3 to trigger cell death. Her team has already found that breast cancer cells die more quickly when they are exposed to both vitamin A compounds and chemotherapy than when they are exposed to either one alone. These findings could lead to a new approach for treating breast cancer.

Regulation of Estrogen Response by Corepressors.
Breast development is regulated by interactions between hormones and growth factors. The hormone estrogen is one of the most important in this process. It binds to the estrogen receptor, which is regulated by proteins called corepressors. Martin Privalsky, Ph.D., at the University of California, Davis, is investigating chemical interactions between corepressors and other proteins called kinases, and how this affects the estrogen receptor. This research will provide new information on what happens when breast tumors stop responding to the hormonal treatment tamoxifen, and may lead to the development of new breast cancer treatments.

Role of Id-2 in Breast Cancer and its Relationship to Id-1.
For a cancer cell to spread, it has to be able to move from its original site and then grow and divide in a new environment. Pierre-Yves Desprez, Ph.D., at the California Pacific Medical Center Research Institute, San Francisco, is studying two proteins, Id-1 and Id-2 that are produced in normal and cancerous breast cells and are believed to play a role in this process. Desprez and his colleagues found that invasive breast cancer cells in mice and cancer cells in human breast tissue have high levels of Id-1. Dr. Desprez is now investigating the Id-2 protein, which, in contrast to Id-1, is present in low levels in invasive cancer cells. His studies in mice and in human tissue have shown that high levels of Id-2 are more likely to be found in non-aggressive breast cancer cells. This research is providing important insights into how the Id-1 and Id-2 proteins interact and how breast cancer grows and spreads.

Research Initiated in 2003

The CBCRP funded 12 new grants in 2003 to pursue new studies on the pathogenesis of breast cancer. Min-Ying (Lydia) Su, Ph.D., from the University of California, Irvine, was funded to use a magnetic resonance imaging (MRI)-based approach to study angiogenic markers in the earliest phases of breast cancer progression—from hyperplasia through DCIS. Dr. Su and colleagues hope to identify and classify the early cancers that are most likely to undergo formation of new blood vessels (angiogenesis) and progress to life-threatening stages. Verena Kallab, M.D., from the University of California, San Francisco, was awarded a postdoctoral fellowship to study circulating tumor cells (CTCs) from patients with advanced disease. Dr. Kallab will study the cytotoxic effects of breast tumor therapy on CTCs and how key cancer biomarkers on CTCs correspond with the primary tumor. Nadim Jessani, from the Scripps Research Institute, La Jolla, is a graduate student in the lab of CBCRP-funded investigator, Dr. Benjamin Cravatt. Mr. Jessani was awarded a dissertation grant to apply a novel proteomics (i.e., study of the whole protein component profile of a cell or tissue) method to detect the active proteases present in human tumors grown in mice. Proteases, such as metalloproteinases, are key modulators of cell invasion, so knowing the active proteases is much more useful than cataloging them at the gene level.

Tsui-Ting Ching, Ph.D., at the University of California, San Francisco, was funded for a postdoctoral fellowship to study gene variation and gene methylation patterns in cell samples from patients with elevated Her-2. Dr. Ching hopes to identify gene/methylation patterns that underlie Herceptin resistance, since only about 30 percent of patients respond well to this therapy. On the same general theme of drug resistance, Kristiina Vuori, M.D., Ph.D., from The Burnham Institute, La Jolla, was awarded a grant to investigate why about 40 percent of the patients treated with tamoxifen have tumors that don’t respond well to this therapy. Dr. Vuori and colleagues are focusing on a docking protein called Cas that may function as an assembly point for anti-estrogen resistance signaling pathways. Nola Hylton, Ph.D., at the University of California, San Francisco, was funded to study the role of p53 as a regulator of radiation-induced cell death in a mouse cancer model.

Dr. Hylton will be trained in the techniques of basic science and transfer this knowledge to her current expertise in MRI and radiology. Steven Martin, Ph.D., from the University of California, Berkeley, received an award to investigate how an oncogene, called Src, regulates cell signaling though growth factors to influence the breast tissue architecture associated with early malignant events. Loss of cell polarity is a key morphological change in cancer development, and Dr. Martin will study the connection to Src by using specific inhibitors and a 3-D tissue culture system in the laboratory of his colleague, Mina Bissell, Ph.D., at the Lawrence Berkeley National Laboratory.

When breast cancer is detected clinically, it has already been present for many years; first in a pre-cancerous stage and then in small, developmental stages. We have too little information on what is happing at the etiological (i.e, causative) and biological (i.e, genetic) levels during breast cancer progression. Paul Henderson, Ph.D., at the Lawrence Livermore National Laboratory, was funded for a novel approach to measure oxidative damage to DNA. While the ability to repair DNA eventually becomes defective in cancer, cancer etiology is believed to be driven to a large extent by the generation of oxygen-derived free radicals. Using breast cancer cell lines and tumors in animals, Dr. Henderson can feed cells or animals an oxidative
damage-reporting marker for detecting and measuring DNA damage. This approach will enable the measurement of the ability of cells to either develop lesions or repair the damage. In recent years the role of the BRCA1 gene in DNA repair has become better defined, but we still need more information on its multiple roles in coordinating the cell cycle, protein degradation, and gene regulation. Quan Zhu, Ph.D., at the Salk Institute for Biological Studies, La Jolla, is a postdoctoral fellow in the laboratory of Dr. Inder Verma. Dr. Zhu will use new gene expression vectors to enable the many BRCA1 functional domains to be studied independently in a mouse model of breast cancer. A paradox in breast cancer biology and the clinic is why about one third of patients at diagnosis are estrogen receptor negative (ER-). Keon Wook Kang, Ph.D., has been awarded a postdoctoral fellowship to study the ER+ to ER- transformation using a special mouse model in Dr. Eva Lee’s lab at the University of California, Irvine. Another perplexing issue in breast cancer is how to associate DCIS, both for the odds of clinical progression and biologically, to invasive cancer. Ruria Namba, Ph.D., from the University of California, Davis, is funded as a postdoc in the laboratory of Jeffrey Gregg, M.D., to study DCIS-like hyperplasic outgrowths from pre-malignant mouse mammary tumors for the expression of altered genes and biomarkers of breast cancer. Euan Slorach, Ph.D., from the University of California, San Francisco, was awarded a postdoctoral fellowship to study a novel gene called Melb1 which is associated with embryonic and mammary development. Dr. Slorach will study this gene for its role in breast tumor development in the context of knockout mice lacking Melb1.