Pathogenesis: Understanding the Disease

Researchers in breast cancer tumor biology are seeking answers to many key questions. How are breast cancer cells different from normal breast cells? How do breast cancers escape the limits of growth placed on normal cells? What are the critical underlying genetic characteristics for the major types of breast cancer? Why do breast cancer cells fail to respond to therapies and the body's own immune system? How do breast cancers gain a blood supply and spread in the body? These questions are being addressed at the cellular, molecular, and genetic levels using CBCRP funding. The research grants summarized in this section generally employ the modern tools of molecular biology to understand the unique genes and protein interactions that allow breast cancers to grow, progress, and spread in the body.

We divide the pathogenesis priority area into five subtopics:

Research Conclusions

Outbreak—How Cancer Spreads: Angiogenesis, Invasion, and Metastasis

Profiling Serine Protease Activities in Breast Cancer.

The functional molecular activities that allow breast cancer cells to invade other tissues are complex and not well understood. One generally accepted hypothesis is that proteins called proteases play a central role in promoting the aggressive behavior of cancer cells. Proteases are enzymes; they break down other proteins. The identities of the individual proteases involved in breast cancer remain elusive. Benjamin Cravatt, Ph.D., from The Scripps Research Institute, La Jolla, developed a technique to detect and characterize the protease “profile” of cells and bodily fluids. This approach is valuable because it is based on the protease's chemical activity, not just the amount present. Thus far, this research has been promising, because the team has detected previously undiscovered proteases that are active in breast cancer cells, but less or not at all active in normal cells. Some of these proteases are from aggressive, ER-negative cell lines, a type of breast cancer that is difficult to treat. In addition, the protease profiles can be used to tell one cancer subtype from another—for example, skin cancer from breast cancer. This research was published in five articles, with recent ones in Nature Biotechnology 20:805-9 (2002) and Proceedings National Academy of Sciences, USA 99:10335-40 (2002). Dr. Cravatt is continuing this work with additional CBCRP funding. This research could lead to a therapy based on inhibiting the action of selected proteases.

FAS as an Anti-angiogenic Target in the Treatment of Tumor Metastasis.

Tumor endothelial cells, the cells that line tumor blood vessels, are an attractive target for cancer therapy. Drugs delivered through the blood would have access to them, and they are not genetically altered cells, like cancer. Elizabeth Hindmarsh, Ph.D., from The Burnham Institute, La Jolla, began her research under the title of “Analysis of Angiogenic Pathways in Metastatic Breast Cancer.” She was investigating chemical interactions between proteins during the process of tumor blood vessel formation. During the course of these experiments, Dr. Hindmarsh saw another opportunity for research. She shifted her attention to the process of fatty acid metabolism. Endothelial and tumor cells have a fatty acid synthase (FAS) enzyme that plays a role in these cells' growth and survival. A drug that inhibits this enzyme, called Orlistat, is used to treat obesity. Dr. Hindmarsh found that Orlistat inhibited the division of endothelial cells grown in lab cultures. Much lower concentrations of Orlistat were effective on endothelial cells, but not on another type of cell that is part of the supporting framework for tumor cells, the fibroblasts. The CBCRP funded additional support to Lynn Knowles, Ph.D., in the same laboratory to pursue Orlistat's effect on tumor cells.

Too Much Cell Growth: Defective Messages and Internal Signaling

Studies on the Role of the ER-β in Breast Cancer

Estrogen receptors are proteins in cells that bind with the hormone estrogen, which circulates in the blood. Once this bonding takes place, it triggers other chemical reactions in the cell. Breast cancer cells that have the most widelystudied estrogen receptor, ER-α, depend on estrogen for their growth. This type of breast cancer is the most commonly occurring subset of the disease; it grows more slowly than many other types and can be treated with anti-estrogen drugs like tamoxifen. Eli Gilad, Ph.D., at the Lawrence Berkeley National Laboratory, investigated a recently-discovered estrogen receptor, ER-β. He introduced ER-β into breast cancer cells grown in lab cultures. Some of these cells also had ER-α and others did not. ER-β appeared to play an important role in inhibiting the growth of cells lacking the ER-α. In cells that had ER-α, adding ER-β allowed them to grow even without estrogen. Results from these studies were presented as posters at the 2000 and 2002 American Association for Cancer Research meetings.

The Role of the BMK1-MEKK3 Pathway in Breast Cancer.

The search has been on for more than a decade to explain how cell surface growth receptors, such as Her-2 and the EGFR, start a chain of chemical reactions inside breast cancer cells. These chemical reactions activate genes that cause the cells to divide and tumors to grow. MAP kinases are a collection of molecules that are part of these chemical reactions. Ta-Hsiang Chao, Ph.D., from The Scripps Research Institute, La Jolla, working in Jiing-Dwan Lee's lab, investigated how BMK1 (Big MAP Kinase-1) puts cells into the phase where they divide into two. Using a special method (two-hybrid screening in yeast), the team discovered that BMK1 first interacts with another molecule called a growth factor. Next, BMK1 interacts with SGK, a type of molecule called a protein kinase. This process is necessary for the cell to begin to divide and discovering it completes a key missing piece of the puzzle.

Specificity of Ras Signaling in Breast Cancer.

Signaling proteins in cells are functionally a chain of reactions between molecules within cells that eventually trigger growth response and genetic changes. Collectively, Ras is a large family of signaling proteins that relay messages from proteins on the surface of cells that cause cells to divide. In about 30% of human cancers Ras becomes mutated in a way that continuously stimulates cell growth. Human cells contain four Ras proteins with similar structures. Janis Jackson, M.D., from The Scripps Research Institute, La Jolla, studied these four Ras proteins—Ki-Ras4A, Ki-Ras4B, N-Ras and Ha-Ras—and found that each one may participate in different chains of chemical reactions inside the cells, that each may have a different biological role. This research also helps explain why human cancers frequently have mutations that activate KiRas, but not N-Ras or Ha- Ras. This research was published in the J. Biol. Chem. 274:17164-70 (1999).

SBP-1: A Novel Survivin Binding Protein in Breast Cancer.

Survivin is a molecule that is present in the cells of human fetuses. Survivin is not produced in normal adult tissue and is consistently overproduced in breast cancer cells. It inhibits the normal process of cell death. Chemotherapy drugs work by triggering the normal process of cell death, and chemotherapy-resistant breast cancers keep these drugs from working by stopping the normal cell death process. Kazuya Okada, M.D., Ph.D., of The Burnham Institute, La Jolla, discovered a protein that binds to Survivin, SBP-1. He described the molecule-level process that promotes the destruction of the Survivin molecule, involving SBP-1 and other proteins found in cells. This work is an important step toward possibly controlling Survivin to allow cancer cells to better respond to drug treatments.

The Control of Breast Cancer Cell Death.

Daria Mochly-Rosen, Ph.D., from Stanford University, applied expertise and prior research in heart disease to investigate how breast cancer cells overcome the body's restraints that should keep them from growing inappropriately. There are two levels of restraints; first, control mechanisms that keep cells from dividing, and second, controls that cause cells to die. The protein kinase C (PKC) family of enzymes is part of the chain of chemical reactions within cells that controls both division and death, in normal and cancerous cells. Dr. Mochly-Rosen's team found that blocking the action of one type of PKC, called delta-PKC, would accelerate tumor growth in animals, and, under certain conditions, prevent the death of breast cancer cells in lab cultures. The research team plans future research to see whether it is possible to make delta-PKC more active in breast cancer cells, and whether this will stop cell growth or cause cell death. If so, this research could provide the basis for a future therapy.

A Novel Signal Transduction Pathway in Breast Cancer.

The CBCRP has funded several studies of the NF-κ-B pathway, a series of chemical reactions within cells that is involved both in cell death and cell division. This pathway is also involved in many aspects of cell function, including stress, injury and especially the immune response. Although the precise role of NF-κ-B in breast cancer is uncertain, this pathway does allow cancer cells to evade cell death and contributes to cancer cells' resistance to immune attack and drug therapy. Yixue Cao, Ph.D., from the University of California, San Diego, working in the lab of Michael Karin, Ph.D., used mouse genetics to investigate a specific protein necessary for the NF-κ-B chain of chemical reactions to take place. Dr. Cao found that this protein, called IKK-α, was essential for the mammary gland (the mouse equivalent of the breast) to produce milk. However, this protein was not necessary for mammary development. A therapy based on blocking the action of IKK-α might make breast cancer cells more likely to go through the natural process of cell death, and this approach might be relatively free from side effects. This research was published in Cell 107:763- 75 (2001). Dr. Karin has received further funding from the CBCRP to pursue this line of research.

Role of the EphB4 Receptor Tyrosine Kinase in Breast Cancer.

Elena Pasquale, Ph.D., at The Burnham Institute, La Jolla, investigated a molecule called EphB4. EphB4 is present at high levels in breast tumors that grow quickly and spread to other body parts. It is also present in other breast cancer cells and probably plays a role in breast cancer, especially in more aggressive tumors. EphB4 is a receptor protein. Part of its chemical structure is exposed on the surface of the cell, the rest of it inside the cell. The exposed portion combines chemically with a protein called ephrin-B2, and this causes the inner portion to start a chain of chemical reactions within the cell. Ephrin-B2 is necessary for embryos to develop blood vessels and is also present in tumor blood vessels. The research team found that the outer portion of EphB4 on the surface of tumor cells promotes the growth of the tumor's blood vessels, and therefore growth of the tumor. However, when EphB4 combines with ephrin-B2, the resulting chemical reaction within the cells may stop the growth and spread of the tumor. There fore, a therapy aimed at EphB4 should either stop the cell from producing it, or promote the chain of chemical reactions caused by EphB4 combining chemically with ephrin- B2.

Novel Mechanisms of ErB-2-Mediated Breast Cancer Metastasis.

In approximately 30% of breast cancers, the ErbB-2 (also known as HER-2/neu) gene has mutated. These cases of breast cancer are likely to spread to other body parts and be deadly. Researchers believe the ErbB-2 gene facilitates this spread. Richard Klemke, Ph.D., at The Scripps Research Institute, La Jolla, investigated interactions between the proteins produced by the ErbB-2 gene with other proteins in breast cells. The team found that once the ErbB-2 protein is interacting within a cell, the combining of two other proteins, CAS and Crk, plus a chain of chemical reactions (a signaling pathway) among proteins called ERK are critical for the cells to move and spread to other body parts. The ERK pathway is part of a family of pathways that trigger normal processes, such as inflammation, and also are involved in the transformation of normal cells into cancer. Another protein, the Abl tyrosine kinase, keeps CAS and Crk from combining and thus keeps cells from being able to move. Breast cancer cells that have the ability to move have lower levels of Abl than those that can't move. Dr. Klemke plans to continue this line of research.

Mistakes on the Master Blueprint: Molecular Genetics and Gene Regulation

A Role for RAD51B in Breast Cancer.

Every cell in the human body contains DNA, a “blueprint” for the cell. When cells divide, errors can occur in replication, and DNA can get damaged during normal cell function. DNA repair proteins found naturally within cells are continually removing small sections of DNA and repairing them. RAD51 is a DNA repair protein that works with the hereditary breast cancer genes BRCA1 and 2. Normal BRCA genes prevent tumors. When mutations in the BRCA genes are present, then RAD51 cannot repair DNA and mutations are passed on to the next generation of cells. Joanna Albala, Ph.D., from the Lawrence Livermore National Laboratory, investigated five proteins with structures very similar to RAD51, focusing on one called RAD51B. She found that RAD51B does not interact with the BRCA1 and 2 breast cancer genes. Her research also shed light on how RAD51, RAD51B, and the four other proteins associate and their locations in breast cancer cells. Dr. Albala published four articles on this work, including a recent paper, J. Biol. Chem. 277:8406-11 (2002). In the course of this project, Dr. Albala received a National Cancer Institute Shannon Award to further support her research on the role of RAD51B in breast cancer.

A New Gene Regulation Factor, LMO-4, in Breast Cancer.

Transcription factors are proteins that turn genes on or off. In search of the causes of breast cancer, many researchers became interested in transcription factors involved in normal breast development. Bogi Anderson, Ph.D., at the University of California, Irvine, identified a new transcription factor, LMO-4. In rabbits and guinea pigs, LMO-4 is most abundant in these animals' equivalent of breast epithelial cells (the cells where most breast cancer arises) during mid-pregnancy. Other investigators have shown that LMO-4 is found in a large portion of human breast cancer that has spread to other body parts. The team found several proteins that may also have to be present for LMO-4 to turn genes on or off. One of these proteins is present in higher than normal amounts in breast cancer cells. It is too early to tell whether LMO-4 is an important protein in breast cancer, but it does appear to be involved in normal breast development, and, perhaps, the early stage of the disease.

Searching the Unknown: Novel Breast Cancer Genes

Analysis of a New Human Caspase in Breast Cancer.

Caspases are proteins that split other proteins, causing the normal process of cell death (apoptosis). The growth of cancer is caused by an imbalance between the rates at which cancer cells are produced through cell division and the rate at which they die through apoptosis. Defects in the cell processes that lead to apoptosis allow cancer cells to survive for prolonged periods of times, accumulate genetic errors, and live in a suspended state that permits them to spread to other body parts. Researchers are in the final stages of cataloging all the human caspases, and work is progressing on understanding how caspase activity is held in check until apoptosis starts. Sug Hyung Lee, M.D., Ph.D., at The Burnham Institute, La Jolla, cloned and studied a new human caspase that was initially discovered in mice. Dr. Lee succeeded in isolating the gene that produces this caspase. Dr. Lee also found another gene that produces a protein similar to a caspase, called COP, which may also play a role in breast cancer. This work was published in the J. Bio. Chem. 276:34495-500 (2001). Dr. Lee conducted this research in the laboratory of John Reed, M.D., Ph.D. Dr. Reed is a world leader in apoptosis research, funded through the CBCRP as a Principal Investigator and through fellowships to many researchers in his lab.

Tumor Suppression by Dystroglan in Breast Epithelial Cells.

Normal breast epithelial cells (the cells where most cancers arise) are organized in a single layer, with one side of each cell attached to another type of cell, collectively called the basement membrane. Proteins attach the cells together. The cell-basement membrane interaction helps prevent uncontrolled cell growth. There is considerable evidence that restoring critical attachment functions in the very early stages of breast cancer will reverse the disease. John L. Muschler, Ph.D., of the Lawrence Berkeley National Laboratory, studied a basement membrane protein, called laminin, which interacts with a protein present on the surface of breast cells called dystroglycan. In addition to interacting with laminin, dystroglycan tells the cell to stop growing. Dystroglycan appears to be absent or nonfunctional in breast cancer. Dr. Muschler described two forms of dystroglycan, α and β, on tumor cells. As epithelial cells transform into tumor cells they start producing enzymes that break dystroglycan-α off the surface of epithelial cells. As dystroglycan-α is lost, the cells become unable to attach to basement membrane. If more were known about this dystroglycan process, it could possibly be used to turn cancer cells back into normal ones. Dr. Mushcler is completing this project with CBCRP funding.

A Novel Antigen Associated with Breast Cancer Metastasis.

Jacqueline Testa, Ph.D., at the Sidney Kimmel Cancer Center, San Diego, identified a previously undiscovered protein that plays an important role in the spread of breast cancer cells to other body parts. The team found the protein by creating a molecule called a monoclonal antibody in the lab. This monoclonal antibody, named mAb 41-2, attaches chemically to proteins in cancer cells that are involved in the spread of tumors. In lab experiments, mAb 41-2 blocks the spread of breast cancer cells. The protein to which mAb 41-2 attaches is present at higher levels in breast cancer cells that have the ability to spread than in normal breast cells. This new antibody could be useful in both diagnosis and treatment of breast cancer.

Unraveling the Path to Breast Cancer: Tumor Progression

Are EGF-Receptors Activated by IL-8 in Breast Cancer?

Normal breast epithelial cells, the cells where most cancers arise, are firmly attached to another structure of cells called the basement membrane. As normal cells turn into cancer, they can become mobile, and they stimulate another type of cell from nearby blood vessels, the endothelial cells, to migrate into the tumor mass to form the tumor's blood vessels. Ingrid Schraufstatter, M.D., from the La Jolla Institute for Molecular Medicine, looked at how tumor and endothelial cells are able to move from the standpoint of proteins that stimulate the normal movement of white blood cells in the body. Interleukin-8 (IL-8) is one of these proteins. IL-8 also controls the movement of cancer cells. Dr. Schraufstatter studied the chemical reactions between IL-8 and two other proteins, one found on the surface of epithelial cells called the epidermal growth factor receptor (EGFR) and the second a protein that allows cells to take in IL-8, CXCR2. This portion of the research was not resolved, but the team found that cathepsin B, a type of protein called a protease, is the key connection between IL-8 and the EGFR. Dr. Schraufstatter presented this research at the FASEB and Keystone meetings in 2002. She will continue investigating the best possible combination of IL-8, EGFR, and cathepsin B inhibitors. This could provide the basis of a therapy that would work by keeping tumor and endothelial cells from moving.

Targets of B Cell Infiltrate in Medullary Breast Cancer.

Medullary carcinoma of the breast is a distinct subtype of human breast cancer that is less likely to be fatal than other types of the disease. Scientists have proposed that this is because the body's white blood cells, part of the immune system, move into the tumor. Henrik Ditzel, M.D., Ph.D., at the Scripps Research Institute, La Jolla, studied one type of white blood cell found in medullary carcinoma tissue, the B white blood cells. The research team cloned the cells and found that they homed in on a protein that is not specific to cancer. This previously-discovered protein is called actin; it plays a role in muscle contraction. The team also found that dying medullary carcinoma cells have the actin protein on their surface, which allows the immune system to recognize the cells. The team further found pieces of the actin protein in medullary carcinoma tissue. The pieces of the protein were similar to those seen after they have been split by a cell death enzyme found in another type of white blood cell, the T cell. The results indicate that the immune system's white blood cells keep medullary carcinoma in check by causing the cancer cells to die, possibly with enzymes found in T white blood cells.

Genes Involved in Immortalization of Human Mammary Cells.

Normal cells stop dividing into two after a limited number of cell divisions. Tumor cells acquire immortality, which is the ability to keep dividing into two indefinitely. Martha Stampfer, Ph.D., at Lawrence Berkeley National Laboratory, studied a cancer gene, Raf. When Raf gets turned on in immortal cells, it promotes tumor growth. When it gets turned on in normal cells, it causes them to rapidly lose the ability to divide in two. The research team found that human breast epithelial cells, the type where most cancer arises, must go through a process called conversion to become immortal. The conversion process is triggered by an enzyme that comes from outside the cell, telomerase. After conversion, turning on the Raf gene promotes tumor growth. If the cells have not undergone conversion, turning on Raf keeps them from dividing. The team also found that other types of mouse and human cells stop dividing when Raf is turned on, but the internal chemical reactions involved are different for different types of cells. Understanding how cancer cells acquire immortality may lead to new therapies.

TGF-β Receptor Signaling and Breast Cancer.

TGF-β is a protein inside breast cells. It inhibits the growth of normal cells, but not the growth of breast cancer cells that have the ability to spread to other body parts. Kunxin Luo, Ph.D., of the Lawrence Berkeley National Laboratory, is investigating TGF-β's role in the normal process of breast cells becoming specialized and in the process that changes normal cells to cancer. Dr. Luo discovered two proteins located in the breast cell nucleus, Ski and SnoN, that block TGF-β. Overproduction of the two proteins kept TGF-β from inhibiting cell growth. Both block the action of another type of protein, Smad. Smad proteins activate a gene necessary for TGF-β to inhibit cell growth, so blocking their action also blocks TGF-β. The research team tested normal and cancerous cells for levels of SnoN. They found little or no SnoN in normal cells, very high levels in early tumor cells that didn't have the ability to spread, and moderate levels in tumor cells with the ability to spread. Thus, a high level of SnoN is a sign that cells are becoming cancerous, and SnoN may play a role early in this process. The team plans future experiments to find out if cells that produce high amounts of Ski or SnoN change from normal to cancerous, and if reducing the amount of these proteins turns the cells back to normal.

Research in Progress

Outbreak—How Cancer Spreads: Angiogenesis, Invasion, and Metastasis

Role of MMPs in Breast Tumor Initiation and Aggressiveness.

Metalloproteinases (MMPs) are enzymes that normal cells secrete to perform a variety of normal processes. Breast cancer cells produce more MMPs than normal; this allows tumors to grow and spread. Jimmie Fata, Ph.D., of the Lawrence Berkeley National Laboratory, is investigating how MMPs can cause normal breast epithelial cells (the type of cells where most cancer arises) 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. The team created cells with EMT characteristics, then inhibited the action of MMPs in these cells. This caused the cells to lose the ability to move individually, the way cancer cells spread, but the cells retain their ability to move in sheets, the way cells move when they close a wound. The team is now investigating how MMPs break apart a protein called Ecadherin, and whether this chemical reaction is a crucial step in breast cancer spreading to other body parts. E-cadherin attaches epithelial cells to other cells in breast tissue.

Analysis of Genes Predictive of Breast Cancer Metastasis.

Jeffrey Gregg, M.D., from the University of California, Davis, is following up on an observation about experimental mouse tumors with an enzyme called phosphoinositol kinase 3 (PI-3 kinase). When PI-3 kinase is turned on, the tumors will spread more often to the lung than to other parts of the body. PI-3 kinase works by activating a series of other enzymes and proteins, and the chain of chemical reactions produced is called the P13-K pathway. The research team has examined the action of PI-3 kinase in two lines of mouse mammary tumor cells (mammary tumors in mice are the equivalent of breast tumors in humans). In a type of mammary tumor cells that always spreads to the lung, the activation of the P13-K pathway is necessary. In another cell line that seldom spreads to the lung, proteins that block the P13-K pathway are active. The team has also found a gene and a protein that are directly involved in the cells developing the ability to migrate to the lung.

TGF-β3 and small GTPases in Invasive Breast Cancer.

Vesa Kaartinen, Ph.D., of the Children's Hospital Los Angeles, is investigating the molecule-level interactions that cause breast cancer cells to become able to move to other parts of the body and form new tumors there. The team is trying to find the proteins involved in the EMT process where one type of breast cell, the epithelial cell, becomes more like another, the fibroblast, and becomes more able to move. Epithelial cells are where most breast cancers arise; mouse mammary epithelial cells are the equivalent of human breast epithelial cells. So far, Dr. Kaartinen has found that one type of protein, transforming growth factor (TGF-β3), changes the locations and amounts of two adhesion molecules (integrins) in mouse mammary epithelial cells. The team has also found that a molecule involved in a chain of chemical reactions within these cells, Rac3, plays a role in the growth of mammary epithelial cells and in the formation of cells that are in transition from normal to cancerous.

Lasp-1 Signaling in Breast Carcinoma Cell Invasion/Migration.

Yi Hsing Lin, Ph.D., at the Scripps Research Institute, La Jolla, is investigating the Lasp1 gene. Dr. Lin's research team had previously shown that overproduction of the protein produced by Lasp-1 is linked to breast cancer cells moving to other body parts. In this study, they have found that the normal Lasp-1 protein prevents cells from moving, but that a mutated version, Lasp-1 deltaC, does not. The mutated version has lost the part of the protein structure, called the SH3 domain, that allows normal Lasp-1 to interact with other cell proteins to prevent cells from moving. The information may lead to new drugs that block the spread of cancer.

Smoking Effect on Pulmonary Metastasis from Breast Cancer.

Smokers are more likely to die of breast cancer than nonsmokers, but they don't get breast cancer any more often. Susan Murin, M.D., of the University of California, Davis, injected mice with breast cancer cells, then exposed some to a level of cigarette smoke comparable to that experienced by actively smoking adults. The mice exposed to smoke had more tumors in their lungs, compared to mice not exposed to smoke. When the researchers halted the animals' exposure to smoke at the time the breast cancer cells were injected (a model for a woman who stops smoking when her breast cancer is diagnosed), the mice had fewer tumors in the lung than the mice exposed to smoke. Next, the researchers will investigate how smoke exposure increases the spread of breast cancer to the lungs.

Too Much Cell Growth: Defective Messages and Internal Signaling

Cell Growth Control of Breast Epithelial Cells.

Ulla Knaus, Ph.D., at The Scripps Research Institute, La Jolla, is investigating two proteins that appear to be involved in the growth of normal breast cells. The two proteins are called Rac3 and Rac1. They are turned on by hormones and by proteins (called growth factors) that come from outside the cell. Rac3 is also consistently turned on in tumors, while Rac1 is not; Rac3 may be tricking cells into growing at inappropriate times. Rac3 does not have mutations, so Dr. Knaus is investigating where inside breast cells this protein is attached. Her team introduced fluorescent copies of both proteins into normal human breast cells. Rac1 distributed itself throughout the cell, but Rac3 attached to membranes inside the cell. They found that Rac3 combines chemically with a particular cell structure called Golgi, and found the part of the Rac3 protein that attaches it. They are now looking for proteins that associate with Rac3 and Golgi that may play a role in growth control.

Molecular Characterization of ErbB2 Positive Breast Cancers.

The ErbB2 protein is present at high levels in 20-30% of all breast cancers, and these tumors are more deadly. The role of this protein is complex; fewer than 20% of these tumors respond well to Herceptin, a drug targeted at ErbB2. Richard Neve, Ph.D., of the Buck Institute for Age Research, Novato, is attempting to find a way to better classify ErbB2- positive breast cancers into distinct molecular subtypes, so that effective therapies could be developed for each subtype. So far, the research team has found that a transcription factor (a type of protein) called ESX is also found in cancer cells that are high in ErbB2, and that ESX may play a role in this type of cancer. Results of this phase of the project were published in Oncogene 21:3934-8 (2002).

Mistakes on the Master Blueprint: Molecular Genetics and Gene Regulation

The Role of BRCA1 in Nucleotide Excision DNA Repair.

Mutations in two genes, BRCA1 and BRCA2, are responsible for 5-10% of all breast cancers. Anne-Renee Hartman, M.D., at Stanford University, has found that the normal BRCA1 gene plays a role in part of a cell's normal process of repairing damage to its DNA. The part of the cells repair process is called nucleotide excision repair. Another gene known to suppress tumors, the p53 gene, is involved in this same process. More than 50% of human cancers have a p53 gene that has stopped working. Dr. Hartman found that a normal BRCA1 gene can compensate for a nonworking p53 tumor suppressor gene.

The Functions of BRCA2 in Repairing DNA Damage.

Women with an abnormal version of the BRCA2 gene are more likely to get breast cancer. The protein produced by the normal BRCA2 gene interacts chemically with a protein complex in cells, Rad51. Rad51 is involved in a part of the process of DNA repair called homologous recombination repair. Yi-Ching Lio, Ph.D., at the Lawrence Berkeley National Laboratory, is using molecular biology methods to investigate the normal BRCA2 protein. This research could shed light on why a mutated BRCA2 gene leads to a high number of mutations in tumor genes, and also help scientists understand why cancer cells can still repair their DNA, even after being treated with chemotherapy that damages DNA.

Regulation of the ATR Checkpoint Response in Breast Cancer.

Dawn Yean, Ph.D., at Stanford University, is investigating the ATR gene, which produces the ATR protein, an enzyme found in cells that plays a major role in DNA repair. Dr. Yean is attempting to find out how ATR works on the molecular level. The research team believes ATR is involved in detecting DNA damage. So far, they have shown that ATR binds to DNA, which suggests their hypothesis may be correct. Next, they will search for other chemical reactions in cells that are necessary for ATR to bind with DNA, and further investigate ATR-DNA interaction.

Unraveling the Path to Breast Cancer: Tumor Progression

A Study of the Molecular Heterogeneity of LCIS.

Women who have the breast disease lobular carcinoma in situ (LCIS) have an increased breast cancer risk. However, LCIS may actually be several diseases, and only a subset of them may lead to a high risk for breast cancer. Sanford Barsky, M.D., at the University of California, Los Angeles, compared the genetic profiles of 200 tissue samples of LCIS. The research team has found that genetically, LCIS can be divided into three types. The first type share characteristics in their genes that suggest that they have progressed to invasive cancer. The second type have genetic profiles that suggest that the disease could develop into cancer. The third type shows no obvious differences in genes with normal breast tissue, suggesting that this type may be harmless.

Immortalization of Human Mammary Epithelial Cells by ZNF217.

The ZNF217 gene is present at higher than normal levels in many breast tumor cells, and it allows cells to continue dividing past their normal limits. Paul Yaswen, Ph.D., at the Lawrence Berkeley National Laboratory, has found that higher than normal levels of ZNF217 may support the survival and growth of cells early in the process when they are becoming cancerous, by allowing the cells to continue growing when they would otherwise stop. In later stages, high levels of this gene may make cancer cells resist chemotherapy and radiation therapy. The research team is studying the chemical reactions within cells in which ZNF217 is involved. If this gene is involved in the earliest stages of breast cells turning into cancer, then further studies could lead to a way to prevent the disease. Results from this research were published in the International Journal of Biochemistry and Cell Biology, 34:1382 (2002).

Genes That Modulate Dioxin-Induced Breast Cancer.

While inherited susceptibility to breast cancer accounts for 10- 15% of all cases, the rest are thought to relate to lifestyle and environmental pollutants. 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, is searching for genes that either promote or suppress breast cancer initiated by dioxin. The research team is using two techniques. The first is RHKO, which has been used to discover genes that inhibit tumor growth. RHKO involves inactivating normal breast cell genes, one gene at a time, then looking for cells that, when a certain gene is inactivated, are immune to dioxin-induced cancer. The second is microarrays, a technology that allows a researcher to study thousands of genes at the same time. The team has assembled the necessary technology and is now testing breast cells.

Tumor Suppression by Dystroglycan in Breast Epithelial Cells.

Cells sense their environment through proteins called receptors, which are located on their cell surface. Receptors combine chemically with molecules outside the cell and then start chemical reactions in the cell that regulate cell behavior. John L. Muschler, Ph.D., of the California Pacific Medical Center, San Francisco, is studying a receptor called dystroglycan. Dystroglycan combines chemically with a protein from outside the cell, laminin, which leads to the cell adopting a particular shape and stops the cell's growth. The molecule is nonfunctional in most breast cancers. Restoring dystroglan function in breast cancer cells makes them more like normal breast cells. Dr. Muschler has found that an important portion of the dystroglycan molecule is detached from the cell surface in the majority of breast cancer cells in lab cultures, and this keeps dystroglycan from working. The loss of dystroglycan function is the result of structural changes that occur in the molecule, not genetic mutation. Dystroglycan also does not function well in the presence of competing chemical reactions in the cell that cause the cell to grow.

Overcoming Drug Resistance in Breast Cancer.

Most chemotherapy drugs work by activating mechanisms in cancer cells that cause the cells to “commit suicide.” It has been hypothesized that resistant cancer cells somehow refuse to commit suicide in response to chemotherapeutic drugs. Kristiina Vuori, M.D., Ph.D., at The Burnham Institute, La Jolla, is investigating how this happens. She has previously found that certain molecules that are on the cell surface, known as integrins, block the suicide process in cancer cells. Normally, integrins anchor cells in their place within a tissue. Integrins on cancer cells appear to have lost their normal function and instead, they may aid the cells' movement and spread. In this study, Dr. Vuori's team has shown that integrins are crucial for the development of drug resistance in cancer cells grown in lab cultures. Dr. Vuori's team is also testing anti-integrin monoclonal antibodies for their ability to make chemotherapy drugs more effective against breast tumors in mice. In addition, they are examining this process at the level of a chain of chemical reactions within tumor cells known as PI 3-kinase/Akt pathway. Results of this study were published in Oncogene 20:4995-5004 (2001).

Research Initiated in 2002

Outbreak—How Cancer Spreads: Angiogenesis, Invasion, and Metastasis

Hox Transcriptional Regulation of Angiogenesis.

Audri Charboneau, Ph.D., at the University of California, San Francisco, is investigating two genes called HOX D3 and D10. These genes may control the molecule-level processes in blood vessel cells that allow breast tumors to create the blood supply that allows the tumors to grow and spread.

The Role of Matrix Metalloproteinase 13 in Breast Cancer.

Mikala Egeblad, Ph.D., at the University of California, San Francisco, is studying an enzyme called Matrix Metalloproteinase 13. This enzyme is secreted by a type of breast cell that supports the epithelial cells where cancer arises. The supporting cells do not themselves become cancerous, but the enzyme they secrete may allow cancer cells to grow and spread.

Method to Profile Active Metalloproteases in Breast Cancer.

Arul Joseph, Ph.D., at The Scripps Research Institute, La Jolla, is using a new chemical method to analyze the amount and activity of a family of enzymes in living breast cancer cells growing in lab cultures. The enzymes are called metalloproteases; there is evidence that they play a role in tumor growth and spread.

Identifying Accessible Targets in Human Breast Tumors.

Jan E. Schnitzer, M.D., at the Sidney Kimmel Cancer Center, San Diego, is attempting to discover new proteins in the blood vessels of breast tumors that are not found in normal blood vessels. This research could open up new opportunities to develop a medication that could neutralize these proteins, kill off a tumor's blood vessels, and starve the tumor.

Too Much Cell Growth: Defective Messages and Internal Signaling

Cell-Killing Effect of Orphan Receptor TR3 in Breast Cancer.

Naathalie Bruey-Sedano, Ph.D., at The Burnham Institute, La Jolla, is investigating a protein found in both human and fruit fly cells called TR3. TR3 may have potential as a breast cancer treatment; the research team will investigate what chemical reactions within cells are necessary for TR3 to trigger the normal process of cell death.

Novel Ligands as Probes of Estrogen Receptor Signaling.

Estrogen receptors are proteins on the surface of breast cancer cells that combine chemically with the hormone estrogen. This can start several chains of chemical reactions within the cell, some of which turn on and off various genes. Nicola Clegg, at the University of California, San Francisco, is creating and investigating molecules that combine chemically with estrogen receptors, but cause or prevent only selected parts of the chains of chemical reactions in the cells.

Cyclin E Affects Growth Arrest in Breast Cancer Cells.

Navdeep Dhillon, at the University of California, Davis, is investigating the protein Cyclin E, which is found in abnormally large quantities in some breast cancer cells, to see if Cyclin E allows cancer cells to become resistant to the drug tamoxifen.

A Novel Anti-estrogen Resistance Mechanism in Breast Cancer.

Kathryn R. Ely, Ph.D., at The Burnham Institute, La Jolla, is investigating the interaction of two proteins, Bcar1/ Cas and SHEP2, to see if they play a major role in breast cancer cells becoming resistant to the drug Tamoxifen.

Structure and Function of the Bax Apoptosis Regulator.

Francesca Marassi, Ph.D., at The Burnham Institute, La Jolla, is investigating the molecular structure of a protein found in many cells, including breast cells, called Bax. The team will also investigate how Bax exerts control over the normal process of cell death.

DNA Damage Response Pathways in Breast Cancer Cells.

Beatriz Maroto, Ph.D., at The Scripps Research Institute, La Jolla, is investigating the complex chains of chemical interactions that normally keep cells with DNA damage from multiplying, and also the chemical processes in breast cancer cells that override this DNA damage control.

Regulation of Estrogen Response by Corepressors.

Martin Privalsky, Ph.D., at the University of California, Davis, is investigating chemical interactions between two types of proteins in breast cells, kinases and corepressors. The research team's actions affect the action of the hormone estrogen on both normal and cancerous breast cells.

Analysis of EGFR Transcript Splicing in C. Elegans.

Cheryl Van Buskirk, Ph.D., at the California Institute of Technology, Pasadena, is using nematode worms called C. Elegans to investigate a protein called the EGF receptor. When cells have abnormally large amounts of this protein, they divide excessively and form tumors. Dr. Van Buskirk is investigating whether genes can produce a variant form of this protein that inhibits excessive cell growth.

Mistakes on the Master Blueprint: Molecular Genetics and Gene Regulation

Alterations in the Separase/Securin Balance in Breast Cancer.

Kelly Boatright, at The Burnham Institute, La Jolla, is investigating the possible role of two proteins, called separase and securin, in causing abnormalities found in the chromosomes of breast cancer cells.

Identifying Sources of Genomic Instability in Breast Cancer.

Karlene Cimprich, Ph.D., at Stanford University, will attempt to catalog and discover detailed information about genes that repair damage to DNA in breast epithelial cells, the cells where most breast cancers arise. The team will also identify which of these genes might be worthy of future study because they are involved in breast cancer.

The Detailed Structure of a Model Breast Cancer Genome.

Colin Collins, Ph.D., at the University of California, San Francisco, is testing a new technique, called End Sequence Profiling, that has the potential to identify all the genetic differences between breast cancer cells and normal cells. End Sequence Profiling uses some of the same methods that were used to map the human genome.

Global Gene Regulation by SATB1 in Metastatic Breast Cancer.

Terumi Kohwi-Shigematsu, Ph.D., at Lawrence Berkeley National Laboratory, is investigating SATB1, a protein that turns on or off hundreds of genes in breast cancer cells, and may also be involved in the spread of breast cancer to other parts of the body.

Searching the Unknown: Novel Breast Cancer Genes

Profiling Enzyme Activities in Models of Human Breast Cancer.

Researchers believe that enzymes within breast cancer cells play a role in the cancer spreading to other parts of the body. Benjamin Cravatt, Ph.D., from The Scripps Research Institute, La Jolla, will use a new method he developed to measure the chemical activity of these enzymes, and not just their abundance, in human breast tumors grafted into mice. Dr. Cravatt is building on research he conducted last year with CBCRP funding, where he used this new method successfully to measure the chemical activity of these enzymes in breast cancer cells growing in lab cultures.

Regulation of the Rad1 Checkpoint Complex in Breast Cancer.

Cells are constantly exposed to agents that can damage their DNA, and cells that don't get cancer-causing mutations in their DNA. Checkpoint proteins in cells sense DNA damage and activate processes to repair them. Patrick Lupardus, at Stanford University School of Medicine, is investigating how two checkpoint proteins, ATR and Rad1, interact to start the DNA repair process.

Cloning of Putative Tumor Suppressor Gene on the X Chromosome.

Sergei Malkhosayan, Ph.D., at The Burnham Institute, La Jolla, is searching for a gene that may prevent tumors in its normal form, and allow tumors to grow when it gets mutated. The team is searching an area of DNA known as the q25 region of the X chromosome. More than 50% of breast tumors are missing some DNA from this region, suggesting that they have lost a gene that suppresses tumors.

Locating Novel Breast Cancer Genes Using DNA Microarrays.

Breast cancer cells often have one or more extra copies of a gene found in normal breast cells, or they are missing a normal gene. Cataloguing the extra and missing genes could improve treatment. For example, Herceptin is a medication used to treat breast cancers with excess Her-2/neu genes on the cell surface, and it became most effective after a test for multiple copies of the gene was developed. Jonathon Pollack, M.D., Ph.D., at Stanford University School of Medicine, is using a new technique called DNA microarrays to find extra and missing genes in more than a hundred tumor samples and in 25 types of breast cancer cells grown in lab cultures.

Unraveling the Path to Breast Cancer: Tumor Progression

Role of PTEN in Progression of Ductal Carcinoma In Situ.

Ductal carcinoma in situ (DCIS) is a pre-cancerous condition of the breast. About 25-30% of DCIS cases become cancer that can spread to other body parts. Currently, there's no way to predict whether DCIS will become cancer, so some women needlessly undergo chemotherapy and radiation treatment. Shikha Bose, M.D., at Cedars-Sinai Medical Center, Los Angeles, is investigating the hypothesis that measuring the level of a protein called PTEN in DCIS cells can predict whether they will become cancerous.

Prognostic Value of Ras Activation in Breast Cancer.

Gerry Boss, M.D., and Anne Wallace, M.D., at the University of California, San Diego, are testing 300-400 breast tumor tissue samples for a protein called activated Ras, then following up to see which tumors recurred or spread to other parts of the body. High levels of activated Ras may predict which tumors are more dangerous.

Infinite Expansion of Breast Tumor Samples in Culture.

Research on breast cancer cells growing in lab cultures is limited to about eight types of cells. These cells came decades ago from tumors that had spread to other parts of the body. This makes it hard to investigate genes and proteins present at earlier stages of the disease. Drugs tested against the currently available cells may not work the same way against tumors caught in early stages of breast cancer. Previous attempts to grow more kinds of breast cancer cells in lab cultures have failed. Shanaz Dairkee, Ph.D., at the California Pacific Medical Center Research Institute, San Francisco, is attempting to develop a new method that would allow scientists to grow cells in lab cultures from the majority of breast cancer cases. This could lead to the discovery of new molecules involved at all stages of the disease, and possibly drugs to target these molecules. It could also lead to individualized therapy, where drugs could be tested against a woman's tumor cells before treatment.

Does the BLM Gene Co-Regulate BRCA1 in DNA Damage Response?

The normal form of the BRCA1 gene prevents uncontrolled cell growth, and women with a mutation in this gene are more likely to get breast cancer. Albert Davalos, Ph.D., at Lawrence Berkeley National Laboratory, is investigating another gene called BLM to see if the protein BLM produces interacts with the normal BRCA1 gene to prevent uncontrolled cell growth. The team will also investigate whether turning off the BLM gene may contribute to breast cancer.

Molecular Pathogenesis of Metastatic Breast Cancer.

Robert Debs, M.D., at California Pacific Medical Center Research Institute, San Francisco, is searching for combinations of genes that all work together to allow breast cancer cells to spread to other body parts.

Studies of Telomere Capping Dysfunction in Breast Cancer.

David Gilley, Ph.D., at the Lawrence Berkeley National Laboratory, is studying the structures called telomeres, which are found on the ends of chromosomes, and the dysfunction of telomeres in breast cancer cells. His research team is also investigating proteins associated with telomeres to see if these proteins play a role in breast cancer cells developing the ability to multiply indefinitely.

Fatty Acid Synthase and Breast Cancer Breast Cells.

Lynn Knowles, Ph.D., at The Burnham Institute, La Jolla, is investigating how Orlistat, a drug approved for treating obesity, also inhibits fat production in, and kills, tumor cells. The research team will also figure out how orlistat works and identify genes it controls.

Identification and Prognostic Value of ERβ in Breast Cancer.

Estrogen receptors are molecules in breast cancer cells and other cells that chemically combine with the hormone estrogen. This starts many other chemical changes in the cells. Tamoxifen, a treatment for tumors that depend on estrogen for survival, chemically combines with estrogen receptors to block the action of estrogen. A new estrogen receptor, ERβ, has recently been discovered. Dale Leitman, M.D., Ph.D., at the University of California, San Francisco, is attempting to develop an accurate test to measure the level of ERβ in tumors.

Three-Dimensional Modeling of Breast Cancer Progression.

Carlos Ortiz de Solorzano, Ph.D., at the Lawrence Berkeley National Laboratory, is investigating where in the breast and when in breast development breast cancer is most likely to start. The research team will use mice that have been genetically engineered to develop tumors that mimic a deadly type of breast cancer, erbB2-positive. They will study where in the mouse mammary gland (the mouse equivalent of the breast) the tumors arise, and plot the cell-by-cell presence of key proteins. The end result will be a 3-dimensional “atlas” to visualize the disease.

BRCA-1-Dependent Ubiquitin Ligase Activity in Breast Cancer.

Yan Xia, Ph.D., at The Salk Institute for Biological Studies, La Jolla, is studying the BRCA1 gene. Mutations in this gene predispose women to breast cancer. The normal version of the gene suppresses tumors, but no one knows how. Dr. Xia will try to find out how BRCA1 functions through mechanisms that cause protein degradation.