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 broad sub-topics:

Research Conclusions

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

Understanding Breast Cancer Cell Metastasis to Bone. Breast cancer often spreads to the bone, but there is currently no understanding of the molecular mechanism underlying this process. Researchers have discovered specific interactions between some types of cancer cells released from an original tumor and the cells at the new location in the body to which they spread, but not for cancer that spreads from breast to bone. Sonoko Narisawa, Ph.D., at The Burnham Institute, La Jolla, investigated protein molecules in tissues adjacent to the bone that might allow breast cancer cells that break off from the original tumor and circulate in the blood to attach to the bone. After overcoming many technical hurdles, Dr. Narisawa identified a possible homing protein, called CD59, attached to the surface of cells adjacent to bone. Identifying a protein that allows breast cancer cells to attach to the bone via the blood could lead to treatments to prevent the spread of breast cancer or tests to predict whether a particular tumor would spread to the bone.

Intracellular Signaling by PI3K in Breast Cancer Metastasis. Earl Sawai, Ph.D., from the University of California, Davis, studied critical proteins than might regulate the spread of breast cancer to other body parts. His group used a unique mouse model system to compare human breast tumors that have high and low rates of spreading. The key difference is that the tumors that spread activate a protein that triggers other changes in the cell. This protein is called phosphoinisitol-3-kinase (PI3K). Dr. Sawai found that a number of other proteins associated with the spread of breast tumors appeared to increase in amount in the PI3K-activated tumors. This data provides the groundwork for future studies to establish the proteins involved in the spread of breast tumors.

Angiogenesis

A number of studies in this section deal with angiogenesis. When tumors grow larger than 1-2 millimeters, they can no longer survive with the blood vessels that feed surrounding tissue. To grow, the tumors need to hijack the body's blood supply and generate their own blood vessels, a process known as angiogenesis. Angiogenesis is also a key event in tumors developing the ability to spread to other parts of the body (metastasis). Researchers are investigating the cell-level biochemical processes involved in cancer forming new blood vessels, many of which are not yet understood. The eventual goal is to develop treatments that shrink tumors and prevent their spread by blocking angiogenesis.

Metalloproteinases (MMPs)

A group of enzymes, called matrix metalloproteinases (MMPs), play a role in normal tissue development. They are also involved in inflammation, degradation of bone and joint, autoimmune disease, and the invasive migration of cancer cells to other body parts. MMPs are important for angiogenesis, the process where tumors develop their own blood supply, which tumors must do to grow beyond a certain size. MMPs promote angiogenesis because they allow normal body endothelial cells to invade the tumor tissue and form both blood and lymphatic vessels. Over 20 types of MMPs are known. Some are secreted from cells and others remain at the cell surface. In all cases, MMPs work by digesting proteins in the cell's immediate environment, the extracellular matrix. Normally, the extracellular matrix maintains cell and tissue structure and restricts cell movements. Because MMPs are outside the cell, they might more easily be inhibited by drugs than the breast cancer proteins and genes within cells. Drugs to inhibit MMPs, such as Marimastat (British Biotechnology) and BMS-275291 (Bristol-Myers Squibb), are being tested in humans to see if they block cancer progression and angiogenesis. This therapeutic approach does not directly kill cancer cells, but can shrink tumors and is attractive in combination therapy with other drugs. In other clinical applications, MMPs are potential biomarkers of breast cancer for early detection. They, or their digested fragments, are released into the blood and can be detected in plasma and urine. In addition, the action of MMPs in digesting proteins in the extracellular matrix leaves behind a ‘signature’ of digested matrix proteins, which might be detected in biopsy. Researchers are investigating whether this ‘digested’ ECM might cause less aggressive tumor cells to become more active. Thus, research into MMPs holds potential for detection, diagnosis, and treatment of breast cancer.

Leukocyte Recruitment, Angiogenesis, and Breast Cancer. Pragada Sriramarao, Ph.D., from the La Jolla Institute for Molecular Medicine, investigated how immune cells attach to blood vessels within tumors, which may be part of the process the immune system uses to shrink tumors. Dr. Sriramarao's team grew tumors just below the skin of mice and used a direct visualization procedure called intravital microscopy to observe immune cells (white blood cells) attaching to the cells that line tumor blood vessels. When breast tumors are growing, they secrete a variety of proteins to stimulate the process of blood vessel formation (angiogenesis). Dr. Sriramarao found, first, that tumors produced high levels of proteins that encourage the formation of new blood vessels and interfere with immune cells attaching to tumor blood vessels. Second, injecting two soluble cytokines (kinds of proteins that cells secrete) into blood vessels near tumors increased immune cells' ability to attach to the blood vessels. Finally, when cells lining the blood vessels had proteins on their surface, called selectins, that were exposed to the blood, more immune cells attached to the blood vessels. This study illustrates the fine-tuned molecular regulation in tumor blood vessels, and suggests that both blocking a tumor's ability to form blood vessels and increasing proteins that help immune cells attach to tumor blood vessels would potentially allow a better immune response to breast cancer.

Breast Cancer Cell Binding to the Endothelium. Brunhilde Felding-Habermann, Ph.D., at The Scripps Research Institute, La Jolla, investigated how breast cancer spreads to organs such as the bone, lungs, liver, and brain. Once cells break away from the original tumor, they spread through the lymph system and bloodstream. Dr. Felding-Habermann studied an integrin adhesion receptor, a substance found in both normal breast cells and cancer cells. Integrin adhesion receptors help keep normal cells in place, but they play a role in the spread of breast cancer cells. The research team found that an integrin adhesion receptor found on breast cancer cells, called ávâ3, exists in both an activated and a de-activated state. Activated ávâ3 allows the cells to leave the bloodstream and attach themselves to the endothelial cells that line the target organs. Activated ávâ3 allows the cell to invade the new organ more effectively by cooperating with an enzyme, MMP-9, which breast cancer cells produce in order to digest the extracellular matrix, the structure that would restrict movement in their immediate environment. This research identifies activated ávâ3 as a marker for breast cancer cells that can spread and invade other organs. This information may lead to ways to test whether a tumor is likely to spread. Results from this research were published in the Proceedings of the National Academy of Sciences, USA 98:1853-8 (2001).

Role of Chemokine Receptors in Breast Cancer Metastasis. Brett Premack, Ph.D., at the University of California, Los Angeles, surveyed existing breast cancer cell lines for the presence of chemokine receptors. Chemokines are a family of fairly small proteins that are often secreted from injured tissue, inflamed tissue, or some white blood cells. White blood cells have proteins on their surface that combine with chemokines; these proteins are chemokine receptors. When the chemokines combine with the receptors, they play a role in white blood cells moving and attaching themselves to other cells, for example, in the processes of inflammation and autoimmune diseases. Dr. Premack's premise was that chemokines might play a similar role in breast cancer cells being able to spread to other parts of the body. He surveyed 13 different chemokines and receptors in eight breast cancer cell lines, and found that a specific chemokine receptor, CxCR4, was present, but only in a small percentage of the cells in a given population. He used a technique called calcium signaling assays to confirm that CxCR4 does work with a chemokine. Unfortunately, Dr. Premack was unable to clearly associate the presence of CxCR4 with differences in the breast cancer cells' ability to invade other body parts. Continued work in this topic is useful, because drugs being developed to address immune disorders might someday find an application for treating breast cancer.

Spatial Control of Matrix Proteolysis in Breast Cancer. Alex Strongin, Ph.D., from The Burnham Institute, La Jolla, studied molecular interactions that occur when breast cancer cells are migrating to other parts of the body. The goal is to find ways to keep the cells from migrating by interrupting these molecular interactions. The team studied interactions between two types of proteins, metalloproteinases, and adhesion receptors. Since the interactions between these proteins occur on the surface of cells, it may be easier to interrupt them than to block molecular processes that occur within cells. Dr. Strongin's team identified molecular processes involved in activating metalloproteinases, such as MT1-MMP and metalloproteinase-2 (MMP-2), on breast cancer cells. The team characterized the chemical interaction that MT1- MMP uses to make an adhesion receptor, the integrin aV chain, work more efficiently to allow cells to move and attach to new places in the body. The team also found a protein, gC1qR/p33, that acts like a “chaperone” for MT1-MMP. Several publications were supported by this funding, including one in The Journal of Biological Chemistry (2001) 276(28):25705-14 and another in Experimental Cell Research (2001) 263(2):209-23.

A Novel Inhibitor of Breast Tumor Angiogenesis. YingQing Sun, Ph.D., from The Burnham Institute, La Jolla, studied molecular-level interactions of a protein—plasminogen kringle 5 (K5)—that inhibits breast cancer cells' ability to form new blood vessels. Cancer cells need to develop this ability to form their own blood vessels (angiogenesis) in order to spread in the body. Dr. Sun discovered two receptors (proteins that bind with K5) on the surface of cells that line blood vessels. Further research on this topic could lead to a novel way to block blood vessel formation in tumors.

The Shp-2 Tyrosine Phosphatase in Breast Tumor Migration. YingQing Sun, Ph.D., from The Burnham Institute, La Jolla, investigated Shp2, a type of enzyme called a kinase. Kinases are found in human cells; they transmit signals that come from the cell surface to control cell functions, including gene activity. Shp-2 plays a pivotal role in triggering the production of metalloproteinase enzymes (MMPs). Breast cancer cells secrete MMPs to facilitate moving to other parts of the body. Using biochemical methods, Dr. Sun identified several proteins in cancer cells that Shp-2 may act on to trigger the molecule-level changes that lead to production of MMPs.

Identifying Breast Cancer Targets for Protease Inhibitors. Yongsheng Liu, Ph.D., from the Scripps Research Institute, La Jolla, used a novel method for detecting proteases, which are proteins breast cancer cells secrete that break down other proteins and allow the cells to move. Up to 2% of the human genes revealed through the Human Genome Project are predicted to be proteases, and only a fraction of them have known functions. Dr. Liu was looking for proteases that can be blocked by serpins. Serpins are types of protease inhibitors, which are proteins produced in the human body that block the action of proteases. Serpins are the principle types of protease inhibitors found in the plasma portion of human blood. Other research shows that serpins inhibit breast cancer cells' ability to invade other body parts. Discovering which proteases serpins act against could lead to new targets for drug therapy. Before resigning from this CBCRP-funded research to take another position, Dr. Liu was able to confirm that the novel method for detecting proteases works.

The Role of IL-8 and its Receptors in Angiogenesis. Interleukin-8 (IL-8) is a substance produced in the human body known for its role in inflammatory diseases, where it attracts white blood cells into an area of tissue injury. IL-8 is also found at high levels in breast cancer cells, especially in more deadly cases of breast cancer. Ingrid Schraufstatter, M.D., from the La Jolla Institute for Molecular Medicine, studied IL-8 in relation to the primary type of normal blood vessel cell that responds to the chemicals tumors secrete to grow their own blood supply. Her research team found that when they inhibited IL-8 receptors (proteins that combine with IL-8) on the surface of normal blood vessels cells, it stopped processes cancer cells need to form new blood vessels. Dr. Schraufstatter also partially traced the biochemical processes IL-8 triggers inside normal blood vessel cells. These experiments predict that blocking the biochemical process IL-8 initiates in normal blood vessel cells has potential as a treatment for breast cancer. Dr. Schraufstatter presented her findings at the 2002 Federation of American Societies for Experimental Biology (FASEB) meeting.

How Does Endostatin Inhibit Breast Cancer Angiogenesis? Endostatin is an anti-angiogenic protein (a protein that inhibits the formation of blood vessels). A large amount of research is being done on endostatin, because it eliminates cancer in mice without side effects or creating tumor resistance. However, researchers don't understand very clearly how it works. Kristiina Vuori, M.D., Ph.D., from The Burnham Institute, La Jolla, investigated how endostatin affects the ability of the cells that line blood vessels to adhere to other cells. Dr. Vuori and collaborators reported (Proceedings National Academy Sciences, USA 2001 98(3):1024-9) that endostatin binds to two classes of endothelial integrin adhesion receptors. These receptors are proteins found on the surface of cells that line blood vessels. Their main function is to attach the cells to surrounding cells that provide structure. This research supports the concept of targeting blood vessels as a promising strategy for eliminating breast cancer.

Cell Adhesion and Drug Resistance in Breast Cancer. Breast cancer cells can sometimes avoid being killed by chemotherapy, and researchers are studying the many biological ways that this resistance develops. Kristiina Vuori, M.D., Ph.D., from The Burnham Institute, La Jolla, confirmed her hypothesis that one key element of drug resistance has to do with the way breast cancer cells are attached to the scaffold of supportive cells that surrounds them. Chemotherapy works by starting a natural process of cell death called apoptosis. Dr. Vuori found that when breast cancer cells are attached to surrounding cells via proteins called β1 integrin adhesion receptors, it reduces the effectiveness of the chemotherapy drugs vincristine and paclitaxel. The biochemical reactions leading to cell death that these drugs start are greatly reduced. How can this be corrected? Dr. Vuori has identified a series of biochemical reactions—called signaling pathways—that link attachment to surrounding cells via β1 integrins to inhibition of the biochemical processes involved in cell death. If the biochemical process that leads to cell death can be restored by drugs, chemotherapy could successfully treat these breast cancers. Results from this research were published in Oncogene (2001) 20:4995-5004. The CBCRP funded Dr. Vuori during 2001 to pursue this work in greater detail.

Proteins

Many researchers on these pages are investigating proteins. Proteins are complex, highly varied molecules that interact chemically within the cells of the body. In every process a cell goes through, proteins are involved. One protein within a cell can break or create a chemical bond in the molecular structure of a second protein, changing the second protein in a way that causes it to change other proteins. Eventually, these changes set off a cascade of chemical reactions that causes the cell itself to change. Genes cause cells to have the characteristics they have by making proteins. Some proteins can turn genes on and off. By studying proteins and their actions in breast cancer cells, researchers are hoping to find new ways to stop or prevent the disease.

Targeting Breast Cancer Blood Vessels. Jan Schnitzer, M.D., from the Sidney Kimmel Cancer Center, San Diego, discovered proteins on the surface of blood vessels cells in breast tumors that are not found on the surface of normal blood vessel cells. The research team made antibodies to these proteins. When injected into the bloodstream, these antibodies had the ability to specifically target tumor tissue. The long-term plan is to use these proteins as targets for drug therapy. Current attempts to use antibodies to deliver anti-cancer drugs directly to breast tumors have met with limited success. When these antibody-delivered drugs are injected into the bloodstream, they don't get across the blood vessel wall to attack the tumor. Cells lining tumor blood vessels are more accessible. Drugs targeted to these cells with antibodies could be used to treat breast cancer by choking off the tumor blood supply. Interestingly, the preliminary results of this research indicate that a naturally occurring cell nutrient and receptor recycling structure in cells called the caveolae that line the blood vessels might prove the most promising avenue for targeting the tumor blood vessels.

Too Much Cell Growth: Defective Messages and Internal Signaling

Novel Binding Functions of Mutant p53 in Breast Cancer Cells. Koji Itahana, Ph.D., from the Lawrence Berkeley National Laboratory, studied p53 tumor suppressor pathway dysfunctions in breast cancer. The p53 pathway is a series of interactions among proteins in cells that leads to cell death after chemotherapy or radiation treatments. Only about 25% of breast cancers appear to have either mutations in or loss of the protein that starts the p53 pathway. Researchers are interested in finding other defects in the proteins involved in the p53 pathway that might also prevent cell death. Dr. Itahana failed to find what he was originally searching for—mutations that made p53 more effective. However, his work led him to study a protein that appears to control the abundance of other proteins in the p53 pathway, but not p53 itself. This protein is a transcription factor, a protein that binds to DNA and controls gene activity. Dr. Itahana found a subtle variation in this protein in some breast cancer cells, but not in normal cells. When this slightly different protein binds to a gene that generates one of the proteins involved in the p53 pathway, the gene generates a slightly aberrant protein. Dr. Itahana plans future research to sort out the role of this process in breast cancer.

COX-2 and Apoptosis Regulation in Breast Cancer. Normal cells have a lifespan; they die naturally and are replaced by new cells. The pre-programmed death process is carried out through a series of moleculelevel interactions in the cell, and is known as apoptosis. Defects in apoptosis interactions allow cancer cells to survive beyond their normal lifespan, to spread to other body parts, and make them hard to kill with chemotherapy or radiation. So it's important to understand on the molecular level how tumors evade preprogrammed cell death. Youngsoo Kim, Ph.D., from The Burnham Institute, La Jolla, began his research by investigating a prostaglandin. Prostaglandins are chemical compounds produced in the body; they are involved in smooth muscle contraction, blood clotting, inflammation, and many other functions. Dr. Kim tested whether the prostaglandin produced by the COX-2 gene inhibits apoptosis. It didn't, but another prostaglandin, PGJ2, appeared to make breast cancer cells more likely to undergo apoptosis. PGJ2 influenced a protein in or on the nucleus of cells called PPARγ. PPARγ combines with other substances produced in the body and also certain drugs, which causes the breakdown of another protein in the cell called FLIP. Loss of FLIP, in turn, allows breast cancer cells to respond to the apoptosis process. These findings solve part of the puzzle of why TRAIL (a protein found in many cells in the body, Tumor Necrosis Factor-related apoptosis-inducing ligand) causes apoptosis in cancer cells. Excess FLIP protects cancer cells from TRAIL-caused death. Knowing the key pathways within cancer cells responsible for cell death provides an opportunity to explore ways of enhancing the ability of chemotherapy drugs and other agents in treating the disease.

Searching the Unknown: Novel Breast Cancer Genes Role of DNA Damage Response Gene in Breast Cancer. Damage to DNA is widely considered to be a dominant force in the generation of cancer. Understanding how cells prevent DNA damage can lead to methods to prevent cancer. Eric J. Brown, Ph.D., of the California Institute of Technology, Pasadena, investigated the ATR gene, which may play a role in either DNA repair or in preventing cells that have cancer-like properties from multiplying. His team created genetically-altered mouse cells and a recombinant virus that could eliminate the ATR from the cells. Their experiments showed that cells need ATR to grow and multiply, but not to function in a stable manner. This study suggests that a chemotherapy that inhibits ATR may prevent tumors from growing. In future studies, Dr. Brown plans to investigate whether ATR has any effect on genes known to be involved in the development of breast cancer, including BRCA1, Chk1, Chk2, and p53. A publication based on this study appeared in Genes and Development 14(4):397-402.

Chromatin Regulation of Breast Cancer Cell Senescence. Every cell's chromosomes contain DNA bundled with proteins to form a complex known as chromatin. Some chromatin proteins protect chromosomes from damage that accumulates as cells age. Paul Kaufman, Ph.D., at the Lawrence Berkeley National Laboratory, is exploring how damage occurs to DNA in human cells during cellular aging, and how chromatin proteins might delay this process. His team studied a protein called Chromatin Assembly Factor, or CAF-1. CAF-1 is responsible for assembling chromatin, and Dr. Kaufman attempted to examine its role in living cells by blocking its function. It was difficult to do at first, but the team succeeded by using a fragment of CAF-1 to inhibit it. Continuous blocking of CAF-1 arrests cell growth. With more research, this information might lead to a treatment that inhibits breast cancer cell division, or reveal how chromatin changes as normal breast cells age, making them more susceptible to cancer.

DNA and Cancer

All cells in the human body contain an “operations manual” made up of DNA molecules. The information in the manual is divided into “chapters” called genes. Genes cause the cells to make proteins that tell the cell how to function. Changes in DNA, called mutations, can be subtractions from, additions to, or rearrangements of the structure of the original DNA molecule. Mutations can happen in the course of cell division, or in the process of normal cell function. Cells can repair most mutations. Cells divide to replace worn-out cells, and the DNA replicates itself to pass on the same traits to the two new cells. If a cell divides before a mutation is repaired, both new cells will have the mutation. Most mutations can't make a cell cancerous. Proteins produced by a very small proportion of cell's genes regulate cell growth and division. A series of mutations in these genes can eventually lead to cancer. Buildup of these mutations may take years. Cells that divide frequently, such as breast cells, are at higher risk for mutations.

Molecular Structure of BAG-1: A New Protein in Breast Cancer. Kathryn Ely, Ph.D., at The Burnham Institute, La Jolla, studied the threedimensional structure of BAG-1, a key protein that regulates apoptosis, the process of programmed cell death. The level of BAG-1 is elevated in breast cancers. It promotes tumor growth and the spread of breast cancer to other parts of the body, which makes breast tumors resistant to anti-cancer drugs such as tamoxifen. Dr. Ely generated a molecular image of BAG-1 using nuclear magnetic resonance (NMR). She found the key structural elements that allow BAG-1 to interact with another protein, called Hsp70. How BAG-1 and Hsp70 function to stabilize each other will take much more research. However, this type of work is critical to explain the mechanism of breast cancer resistance to antiestrogen drugs such as tamoxifen. This research also points the way toward strategies to overcome treatments that fail women with breast cancer. Dr. Ely's research was published in Nature Structural Biology 4:349-352 (2001). The CBCRP funded continued research on this topic in 2001.

Characterization of hAG-2 and Its Role in Breast Cancer. When surgeons remove breast tumors or perform biopsies to get samples of tumors, the tissue gets evaluated for several characteristics that help determine the prognosis and best treatment. One of the most useful of the characteristics is the presence of proteins called estrogen receptors (ER), because tumors that contain this protein often respond well to anti-estrogen drug treatment. ERpositive breast cancer is also the most common form. Devon Thompson, Ph.D., from Stanford University, Palo Alto, studied a protein, hAG-2, which was found together with ER in 79% of the tumors she examined. She was not able to determine whether hAG-2 plays any role in breast development or whether it responds to hormones. Dr. Thompson and colleagues published related work on estrogen-responsive genes in Cancer Research 60:6367-75 (2000). Even though ER-positive tumors respond to therapy, there is the need for new drug targets to enhance treatment options and deal with the problem of resistance to the drug tamoxifen.

Suppressor Genes of Breast Cancer. Research over the past two decades has shown that cancer is caused by changes in genes within cells, thus leading to the theory that cancer is a genetic disorder. Many genes present in normal cells are absent in cancer cells. This suggests that there are a large number of genes that suppress the growth of tumors, and that their absence allows genes that promote tumor growth to be active. Unfortunately, to date only a handful of tumor suppressors have been identified. Shi Huang, Ph.D., of The Burnham Institute, La Jolla, studied a group of genes which are members of a family of genes, the PR family. Other known members of this gene family suppress cancer formation and growth of new cells. Dr. Huang investigated three genes from this family, PFM4, 7, and 11. These genes produce proteins that inactivate other genes. When the genes are absent from a cell's DNA, other genes that are normally inactive would be expected to be active. Dr. Huang's team examined the DNA in tissue samples from 40 primary breast cancers for changes in the three PMF genes. PMF4 was either absent or altered in only about 20% of the samples. Dr. Huang plans to continue investigating these genes to see whether other reasons might account for the genetic “silencing” of PFM4.

Unraveling the Path to Breast Cancer: Tumor Progression

Id-1 Expression During Breast Cancer Progression. Pierre-Yves Desprez, Ph.D., at the California Pacific Medical Center Research Institute, San Francisco, investigated Id-1, a type of protein called a transcription factor, in various stages of breast cancer. Using tissue samples from tumor biopsies, he found that the Id-1 protein was mostly absent in samples taken from women diagnosed with DCIS (non-cancerous lesions that may later develop into cancer) and Grade 1 tumors (which do not spread). In contrast, the Id-1 protein was present in substantial amounts in more than half of the Grade 2 and 3 tumors, which are more likely to spread. Other work from Dr. Desprez's laboratory shows that Id-1 plays a key role in allowing breast cells to activate genes for invasive metalloproteinase enzymes. Cells release these enzymes to pave the way to migrate in the body. Thus, blocking Id-1 would be useful in preventing the spread of breast cancer, and knowing whether Id-1 was present could provide information about whether a breast tumor is likely to spread. The CBCRP has funded Dr. Desprez in 2001 to continue this work and examine an additional protein, Id-2, for its role in breast cancer.

Research in Progress

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

Profiling Serine Protease Activities in Breast Cancer. Benjamin Cravatt, Ph.D., at The Scripps Research Institute, La Jolla, is using proteomics, the simultaneous analysis of the complete protein content of given cell or tissue, to study breast cancer. He has developed a special technique to measure active protease enzymes. This approach is an improvement over methods that only measure the presence of genes and proteins and do not actually determine the associated enzyme activity. Breast cancer cells use these proteases to break down proteins in their environment and move to other parts of the body. Dr. Cravatt is developing key information on forms of breast cancer that do not depend on the hormone estrogen for growth (ER-negative breast cancer). ER-negative breast cancer cells frequently utilize a protease called urokinase to invade other cells. It is important to develop more information on ER-negative breast cancer, since this form is not well controlled by chemotherapy medications such as tamoxifen. Dr. Cravatt's methods were published in Biochemistry (2001) 40:4005-15.

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 increases the tumor blood supply and 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 is associated with aggressive breast cancers. Dr. Fata is studying the process by which MMPs cause EMT on the cell and molecule level, as well as looking at what happens on the cell and molecule level as a result of this process.

Too Much Cell Growth: Defective Messages and Internal Signaling

A Novel Signal Transduction Pathway in Breast Cancer. Yixue Cao, Ph.D., from the University of California, San Diego, showed that a protein called IKK-alpha is critical for mammary gland development. Dr. Cao developed mice with a specific mutation in the gene that produces the IKK-alpha protein. These mice are normal, except that they fail to produce milk. The next step is to find out whether this mutation in the IKK-alpha gene suppresses tumor growth in mice. This preliminary work suggests that a drug that works by blocking IKKalpha will not cause side effects in other organs. For 2001, the CBCRP funded a much larger grant to Dr. Cao's mentor, Dr. Michael Karin, to expand these studies. (See the “Biology of the Normal Breast” section of this annual report, “Research Initiated in 2001.”)

Anti-E-Cadherin Apoptosis of Inflammatory Breast Carcinoma. Inflammatory breast carcinoma is one of the most deadly forms of breast cancer. Mary Alpaugh, Ph.D., at the University of California, Los Angeles, and colleagues investigated a protein, E-cadherin, that normal breast cells produce and which allows the cells to attach in layers to other cells. The research team confirmed that E-cadherin promotes the ability of inflammatory breast cancer cells to spread locally in the breast. This finding challenges the current thinking about the role of E-cadherin, which has maintained that if normal breast cells lose the ability to produce E-cadherin, they become cancer cells. Results from this project were published in Cancer Research 61:5231-5241 (2001).

Studies on the Role of the ER-beta in Breast Cancer. Estrogen receptors are proteins on or in cells that chemically bond 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 widely-studied estrogen receptor, ER-alpha, 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, is studying a recently-discovered estrogen receptor, ER-beta. ER-beta may be involved in molecular interactions inside breast cancer cells with both ER-alpha and a gene that promotes more deadly forms of cancer, Her-2. Dr. Gilad has found that ER-beta will promote tumor growth even in the absence of estrogen. Surprisingly, ER-beta inhibits tumor growth when it interacts chemically with Her-2. This line of research will contribute to treatment decisions, especially for women with breast cancer that is resistant to treatment with antiestrogen drugs. Dr. Gilad works in the laboratory of Ruth Lupu, Ph.D., who was funded by the CBCRP from 1997—1999 to develop key background on this topic.

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. In future experiments, they will try to find the particular cell structure that attaches Rac3. They also found that several growth-stimulating substances that activate Rac1 don't activate Rac3.

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 in two to replace worn-out cells, the DNA also replicates itself. Errors can occur in replication, and DNA can get damaged during normal cell function. So special DNA repair proteins found naturally within cells are continually removing small sections of DNA and correcting the damage. Joanna Albala, Ph.D., from the Lawrence Livermore National Laboratory, is studying a DNA repair protein, called RAD51, 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. Dr. Albala is investigating which of the five RAD51 proteins form the active complexes in breast cancer cells. Part of this research was published in Molecular and Cellular Biology 17:6476-82 (2000).

DNA Packaging Defects in Breast Cancer. Terumi Kohwi-Shigematsu, Ph.D., from the Lawrence Berkeley National Laboratory, is continuing to study ways that the DNA of breast cancer cells differs from that of normal cells. She has identified a protein called poly (ADP-ribose) polymerase (PARP) that binds to a specific stretch of DNA, the matrix attachment regions (MARs). PARP plays a role in DNA repair. Dr. Kohwi-Shigematsu's hypothesis is that breast cancer cells produce PARPs at higher levels than normal cells, and that the PARPs organize the cell's DNA to allow it to maintain its growth rate and invade other cells. Dr. Kohwi-Shigematsu is comparing cells with high and low levels of PARPs to see how PARPs affect the action of other genes in these cells. She is using microarray technology, a technique that allows her to simultaneously check 20,000 genes, so as to find the 'needle in the haystack' of possible genes affected by the PARP variation. She will also use mice that have been genetically engineered to spontaneously form breast cancer that spreads to the lung, and find out if the cancer will still grow if these mice are further engineered so that they cannot produce PARP. A publication based on this research appeared in the Journal of Cellular Biochemistry 35:36-45 (2001).

Searching the Unknown: Novel Breast Cancer Genes

Metastasis Suppressor Genes for Breast Cancer. Stanley Cohen, M.D., at Stanford University, Palo Alto, is attempting to discover novel tumor suppressor genes, which might be a means to inhibit breast cancer growth and prevent its spread to other body parts. The underlying theory is that cancer cells acquire a single mutation in their DNA that de-activates a tumor suppressor gene and then allows the cancer cells to spread. Dr. Cohen is using a technique called Random Homologous Knock Out (RHKO) that allows him to screen a tissue sample for thousands of genes at a time and to quickly identify a target gene. When he finds a possible suppressor gene, he will check to see if it is missing in breast cancer cells, then test to see if inserting it in breast cancer cells makes them behave more like normal cells. As part of this research, Dr. Cohen is working with biotech collaborators to develop a way to externally detect very small tumors that have spread to the lung in living animals, using the fluorescent protein produced by fireflies and a type of video camera. This will allow testing to see if a particular gene causes tumors to spread, without having to sacrifice the animals the animals and interrupt the remainder of the experiment.

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, is comparing the genetic profiles of 200 tissue samples of LCIS. After analyzing 100 samples, Dr. Barsky has tentatively divided LCIS into three types. The first type share characteristics in their genes that suggest that they have progressed to invasive cancer. The second type has 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. Over the coming year, the team will analyze the rest of the 200 tissue samples to see whether the three-tier classification system holds.

TGF-beta Receptor Signaling and Breast Cancer. TGF-beta 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-beta'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-beta. Over-production of the two proteins kept TGF-beta from inhibiting cell growth. 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. Next, the team will examine levels of Smad proteins in normal and cancerous breast cells.

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, the site of most breast tumors. Mary Helen Barcellos-Hoff, Ph.D., at the Lawrence Berkeley National Laboratory, is pursuing 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. Her team is using mouse mammary epithelial cells that lack a gene, p53, that normally suppresses tumors. They predict that when these cells are transplanted into a stromal cell framework that has been exposed to radiation, they will become cancerous more rapidly. They are running the same experiments using epithelial cells with normal p53 genes. Understanding how radiation alters normal mechanisms that stromal cells use to keep epithelial cells from turning cancerous may provide new strategies for augmenting these mechanisms to prevent or reverse cancer.

Research Initiated in 2001

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

Too Much Cell Growth: Defective Messages and Internal Signaling

Research on Metastasis, the Spread of Breast Cancer

Breast cancer spreads through the blood and lymph system to form tumors in other parts of the body. This process is very inefficient. Scientists believe perhaps only one in a million cancer cells released into the blood from a primary tumor will successfully implant in another organ, such as the lung. In addition, in the new organ, the cancer cells often remain quiescent or grow very slowly for years. However, it is the growth of tumor cells in distant organs from the breast that eventually compromises the function of the organ, leads to a critical tumor load (1-2 kg), and overwhelms any therapeutic intervention. Research in metastasis is focusing in the cell surface adhesion receptors and proteases of cancer cells that allow them to migrate, enter the blood/lymph, and exit into other organs. Any breakthroughs that might reduce metastasis and growth in secondary organs is likely to represent a huge advance in reducing deaths from breast cancer.

Mistakes on the Master Blueprint: Molecular Genetics and Gene Regulation

Searching the Unknown: Novel Breast Cancer Genes

Unraveling the Path to Breast Cancer: Tumor Progression