Innovative Treatments: Search for the Cure
Pre-clinical and early clinical studies are the bases for generating radically different ways to treat breast cancer. These CBCRP funded projects are exploring how newly discovered technologies and products can be used to treat breast cancer in more effective, less toxic ways.
Conclusions
Gene therapy and other treatments: new frontiers
Senyon Choe, Ph.D., at The Salk Institute for Biological Studies, completed a 2-year project that investigated Targeting Breast Cancer Using Diphtheria Toxin. This study was designed to develop the potential for diphtheria toxin as a breast cancer therapeutic. Dr. Choe determined the X-ray crystallographic structure of diphtheria toxin under conditions when it is attached to its natural partner, the ‘EGF precursor’ protein. He was able to identify the critical atoms for both diphtheria toxin and the ‘EGF precursor’ that regulate this binding function. In future work, the structure of diphtheria toxin will be modified to make it specific for heregulin, a growth promoting protein found on breast cancer cells. This is possible, because the ‘EGF precursor’ and heregulin are very similar in structure. Thus, the toxic effect of diphtheria toxin could be redirected selectively to breast cancer as a novel therapeutic approach.
Silvia Formenti, M.D., at the University of Southern California, completed an ITaMoCA grant to investigate the utility of radiosurgery for replacing the six weeks of the radiation therapy normally required to treat breast cancer. Radiosurgery delivers a radiation dose in a single session (lasting approximately one hour) that may be biologically equivalent to what is received over six weeks of daily treatments. Dr. Formenti performed a preliminary study to work out the logistics of the technique. Although the optimal radiation dose for killing the tumor still needs to be found, the study was able to demonstrate that radiosurgery is feasible and that patients tolerate the treatment well.
Orhan Nalcioglu, Ph.D., at the University of California, Irvine, completed a 3-year Research Project for the investigation of Improved Drug Delivery in Breast Cancer. This project examined the permeability of the blood vessels in experimental mouse breast tumors using the technique of magnetic resonance imaging (MRI). These tumor vessels are more permeable or ‘leaky’ compared to the normal mi-crovasculature. Dr. Nalcioglu was able to determine the level of leakiness by constructing MRI ‘contrast agents’ of different sizes. Differences in permeability were correlated to the aggressiveness of the tumor with respect to spread in the body. Vasomodulators and other drugs were able to alter the tumor leakiness. These experiments set the stage for the application of MRI for diagnosis in human breast cancer, and to develop ways for the selective delivery of drugs to tumors based on the permeability characteristics of their blood supply.
Ke Shuai, Ph.D., at the University of California, Los Angeles, was funded for 3 years as a New Investigator to study Growth Inhibition of Breast Cancer Cells by Interferons. His initial experiments examined an interferon regulator from fibroblasts, Stat1, but these experiments indicated that this antiproliferative pathway was not operable in breast cancer cells. Next, it was shown that a transcription factor, NF-kB, was activated by interferon, but this did not account for antiproliferative activity. Finally, Dr. Shuai found that specific cytokine signaling factors, called SOCS1 and SOCS3, appeared to protect breast cancer cells from the antiproliferative activity of interferon. Thus, further studies on these factors could lead to possible strategies to make interferon a more effective anti-breast cancer agent.
Hormone and chemotherapy targets: improving today's arsenal
Cells that are drug resistant tend to have a higher overall activity of an enzyme called protein kinase C, but not enough is known about the role of the different forms of protein kinase C (isozymes) in drug resistance. Yuefeng Lu, Ph.D., at the Stanford University, completed one year of a post-doctoral fellowship investigating how the six different protein kinase C isozymes found in breast cancer are involved in the developmentof drug resistance protein kinase C may function either by excluding drugs from cells or by causing the cells to undergo programmed cell death. Different forms of protein kinase C behave differently and even oppositely on these processes. Dr. Lu interfered with the function of specific isozymes and found that certain isozymes (b-PKC) appear to be more involved in protecting cells from programmed cell death than other isozymes (d-PKC and e-PKC). Isozymeselective inhibitors of these protein kinase C isozymes may serve as models for designing agents that would make chemotherapy more effective.
Silvia Formenti, M.D., Peter Danenberg, Ph.D., and Franco Muggia, M.D., at the University of Southern California, have completed a pilot Translational Research Collaboration Award. The purpose of the study was to determine whether there are biological factors in tumors that could be used to predict their response to chemotherapy. In order to perform the study they examined the response of primary tumors to paclitaxel. The researchers found that the paclitaxel unresponsive tumors had very high levels of certain types of b-tubulin (a component of cellular structural proteins) while those that responded well to paclitaxel had the lowest levels. Findings from this pilot study justified a larger translational research project aimed at finding the biological factors that determine the response to paclitaxel and paclitaxel plus radiation. The ultimate aim of these investigations is to spare patients with resistant tumors from exposure to unnecessary toxicity and to optimize the treatment of patients with potentially responsive tumors.
Immune therapy: mobilizing the body's defenses
Many researchers believe that the immune system can successfully deal with cancer in the same way that it deals with viral infections. A logical extension of this theory is that vaccines can be used to combat cancer growth. One hurdle in developing a cancer vaccine is making it general enough to address the high genetic variability between individuals as well as between tumors, but still making it specific enough to kill only tumor cells. Alessandro Sette, Ph.D., and Esteban Celis, M.D.,Ph.D., at Epimmune, Inc., completed an ITaMoCA award to develop a cancer vaccine by training a particular type of immune cell, the cytotoxic T-lymphocyte, to recognize common “selfantigens” in addition to the proteins that are often found in tumors. This approach increases the possibility that the vaccine will be useful for a broad segment of the population. Drs. Sette and Celis were successful in developing cytotoxic T-lymphocytes that could recognize various tumor markers (HER2/neu, CEA, MAGE2/3, and p53) in cells with a variety of “self-antigens” present. This is a hopeful step toward the development of a cancer vaccine.
Research in Progress
New drug design: creative science
Harnessing the power of retinoids (vitamin A) to stop cells from dividing is an avenue of investigation that holds promise for breast cancer therapy. The types of retinoids that have been tested until now have had some undesirable side effects. Magnus Pfahl, Ph.D., at the Sidney Kimmel Cancer Institute, used his ITaMoCA award to test a new class of retinoids that make cancer cells commit suicide. He found that the new retinoids were able to cause breast cancer cells to die within 24 hours of exposure, and that they turned on a different combination of receptors than traditional retinoids. The new retinoids were too potent to use in combination with chemotherapy as was originally hoped; however, these studies did lead to the discovery of a new compound, MX871, that effectively treats advanced estrogen receptornegative cancer in the laboratory. Clinical trials of MX871 are now being developed.
Gene therapy and other treatments: new frontiers
Current breast cancer treatments generally inflict terrible side effects on patients. This is often because of an inability to target the treatment selectively to the cancer. The same issue applies to gene therapy—we need better ways of delivering therapeutic genes to cancer cells. Robert Debs, M.D., at the California Pacific Medical Center, Geraldine Brush Research Institute, is encapsulating his gene therapy agent inside lipid particles to protect them in transit in the blood and deliver them to cancer cells. He is finding that his technique will target the blood vessel lining cells (endothelial cells) in the tumor blood vessels. Many of our funded projects are finding the tumor's blood supply is a better target than the tumor itself. This strategy is being pursued in a Translational Research Collaboration Pilot grant to Marc Shuman, Ph.D., Randall Hawkins, M.D., and Laura Esserman, M.D., at the University of California, San Francisco. This cross-disciplinary group of scientists is developing the clinical potential of a monoclonal antibody against an angiogenesis-stimulating protein vascular endothelial growth factor (VEGF). They are using both an advanced form of magnetic resonance imaging (MRI) and positron emission tomography (PET) to directly visualize the effects of anti-VEGF treatment. Combining new non-invasive detection technology with emerging state-of-the-art therapy is the strength of this project. Finally, Qing Zhou, M.D., at the University of Southern California, is working with his postdoctoral mentor, Francis Markland, Ph.D., to deliver an angiogenesis- inhibiting snake venom protein directly to the tumor blood vessel cells by gene therapy techniques. Their hypothesis is that this approach will disrupt the tumor blood supply. Since the blood vessel cells are not cancer cells, they are unlikely to develop resistance to this treatment.
Breast cancer patients can develop side effects from the removal of the primary tumors. Alternative, less invasive ways of removing the tumor are highly desirable. Boris Rubinsky, Ph.D., at the University of California, Berkeley, finds promise in an alternative to surgery—freezing the primary tumors. Dr. Rubinsky has found that by injecting cultured breast cells or surgical specimens with certain “antifreeze proteins” and then freezing them, he can achieve complete destruction of the target tissue regardless of how far the temperature is lowered. He is currently in the process of testing this approach in animals.
Breast cancer patients suffer severe effects from the presence of tumor cells that have spread in the body. This is especially true in bone, where the cancer cells cause bone loss and elevated calcium levels in the blood. Herve LeCalvez, Ph.D., at The Burnham Institute, is investigating a bone cell protein, called meltrin-a, which is critical for the fusion of cells to form the bone-destroying osteoclasts. He is studying meltrin-a in experimental cell systems with the aim of developing strategies to block its function. This could have applications both for breast cancer and in treating osteoporosis.
Hormone/chemotherapy targets: improving today's arsenal
Several ongoing CBCRP grants are investigating the Her-2 growth factor, which is present on the surface of breast cancer cells in about 30% of patients. A monoclonal antibody against Her-2 was approved for treatment in 1998, but this topic remains an intense area of interest. Cara Marks, Ph.D., of the University of California, San Francisco, is using monoclonal antibodies and X-ray crystallography to study how Her-2 self-associates into a dimer and how this relates to growth signaling within cells. In a different approach to neutralize the Her-2 receptor, Joel Gottesfeld, Ph.D., at the Scripps Research Clinic, is developing a novel method to ‘turn off’ the gene for this protein. He is investigating pyrrole-imidazole polyamides, which are molecules that bind to specific regions chromosomal DNA and can ‘lock’ genes in an inactive state. Dr. Richard Pietras of the University of California, Los Angeles is addressing the problem of estrogen independence in Her-2 expressing cells. He has found that there is cross-communication between Her-2 and estrogen receptors, which provides a biologic basis for the clinical observation of tamoxifen resistance in patients with breast tumors rich in Her-2 receptors. Resistance of these cancer cells to tamoxifen can be reversed by treatment with an antibody that counteracts the ill effects of Her-2.
Chemotherapy treatment remains a common approach for breast cancer, despite the fact that it is ineffective in many cases. Cells become resistant to chemotherapeutic compounds in a variety of ways, and research is underway to understand the basis for this resistance. Daniel Mercola, M.D.,Ph.D., of the Sidney Kimmel Cancer Center, is researching ways to inactivate an intracellular signaling protein, called Jun kinase, in order to restore the sensitivity of cancer cells to the DNA-damaging drug—cisplatin. When Jun kinase becomes inhibited, the cells lose growth potential and become more sensitive to cisplatin.
Immune therapy: mobilizing the body's defenses
Breast cancer does not appear to stimulate as strong an immune response as some other cancers (e.g., melanoma), so research in this topic often examines the ability of breast cancer cells to avoid this natural mechanism of defense. Several different immune cell types can be recruited in order to train the body to recognize cancer cells and destroy them. One cell type, the dendritic cell, is especially good at processing antigens so that the body can see them. Two CBCRP researchers are making progress in taking advantage of this dendritic cell characteristic, Michael Roth, M.D., of the University of California, Los Angeles, targeting Her-2 and Jeffrey Weber, M.D., Ph.D., of the University of Southern California, targeting CEA. They are removing the dendritic cells from patients, exposing them to the antigen of interest and then returning them to the patient. Ideally, this process will allow the immune system to recognize and attack the breast tumors.
Researchers are also using other cell types in the immune system to train the body to see tumor cells. Jeffrey Smith, Ph.D., at The Burnham Institute, is using T cells to target and kill the cancer cells. Dr. Smith is using a process called protein loop grafting, which allows him to change the function of a protein. He is now fusing a part of the T cell that recognizes the T cell receptor to the part that recognizes cancer proteins in order to target the killer cells more specifically to the tumor cells. He has begun to examine whether these new constructs are properly displayed on the surface of immune cells—one of the first steps toward developing an effective immune therapy.
Antibodies are another component of the immune system that CBCRP investigators are using to specifically kill tumor cells. Sherrie Morrison, Ph.D., at the University of California, Los Angeles, has begun to develop a cancer vaccine by fusing an immune cell growth factor, IL2, with the antibodies to CEA or HER-2. She has made several versions of these fused antibodies and is now testing their effectiveness in mice. Jerry Peterson, Ph.D., at The Cancer Research Fund of Contra Costa, has made a novel type of molecule call Ifabs, which are combinations of antibodies and radioactive agents (radioconjugants). He finds that by using parts of the antibodies that target BrE3 and Mc3 proteins as radioconjugants, he is able to target human tumors. Yoko Fujita-Yamaguchi,, Ph.D., of The Beckman Research Institute, is using antibodies to interfere with IGF-I stimulation of tumor cell growth. Her approach is to block IGF-I binding to its receptor by introducing an antibody that gets in its way. They have learned that stable expression of antibodies against the IGF-I receptor inside of cells is probably lethal to cancer cells. Next, the breast cancer cells expressing insoluble antibodies against the IGF-I receptor will be used to test synergistic effects of chemotherapy and anti-growth factor antibodies.
New drug design: creative science
Breast cancer cells grow in response to estrogen. Tamoxifen works by blocking the tumor cells' access to estrogen. In an alternative approach, Masato Tanabe, Ph.D., of SRI, International, is designing a new drug to treat estrogendependent breast cancer by inhibiting the production of estrogen. These drugs inhibit the production of estrone sulfatase. In the first year of his project, Dr. Tanabe has designed several drugs that have shown promise in cell culture and has developed an animal model in which to test the compounds.
The spread of breast cancer (metastasis) is being intensively investigated for opportunities to develop new drug therapies targeting this process. Francis Markland, Jr., Ph.D., at the University of Southern California, continues to develop the potential of a protein from snake venom to inhibit breast cancer metastasis and angiogenesis. Using support from the CBCRP he has purified, cloned, and established the effectiveness of this venom protein in breast cancer animal models. Since it is so difficult to block breast cancer cells in transit in the blood, he is developing his novel drug with the aim of disrupting the blood supply. This approach would be effective both on the primary tumor and places in the body (e.g., bone and lung) where breast cancer commonly spreads. Two other investigators, Renata Pasqualini, Ph.D., at The Burnham Institute, and Joseph Konopelski, Ph.D., at the University of California, Santa Cruz, are working towards developing therapies directed against the proteins that breast cancer cells use for attachment during invasion and metastasis. Dr. Pasqualini is studying a polymeric form of a protein called fibronectin to block tumor angiogen-esis. Dr. Konopelski is using computer modeling to design protein-based derivatives of a fragment of laminin, a cell attachment protein.
Recently Initiated Research
Under this priority issue, CBCRP awarded 12 grants in 1998. In most cases, the funded projects are a link between the basic science laboratory and the clinic. At this stage there is a need for refinement of the mechanisms of action of new treatments and validation of their potential before they are used in humans. These projects make strong use of both animal model systems of breast cancer and clinical samples. There is an increased awareness that more relevant model systems are needed to test potential treatments for breast cancer. This year our awards are clustered in three topic areas;
- metastasis and angiogen-esis,
- immunotherapy and drug delivery approaches, and
- surgery, tumor markers, and drug efficacy.
Angiogenesis has recently emerged as a promising target for attacking breast cancer. The size, invasiveness, and spread (metastasis) of tumors can be limited by treating the local blood supply. The blood supply of tumors is poorly organized or ‘leaky,’ which allows tumor access for new forms of drug delivery and targeting. Francis Markland, Jr., Ph.D., is exploring the delivery and formulation of a novel protein from snake venom that affects the adhesive properties of both endot-helial and breast cancer cells. Similarly, Keith Laderoute, Ph.D., has identified a new drug that disrupts the internal cytoskeleton of cells, and also targets both the blood vessels and breast cancer cells in tumors. Finally, dietary agents could offer more long-term, preventative affects on tumor angiogenesis. Kent Erickson, Ph.D., is investigating conjugated linoleic acid as a potential angiogenesis inhibitor to treat women at risk for developing metastasis following the diagnosis of breast cancer.
Immunotherapy and associated treatment strategies commonly focus on the breast cancer cell oncogene Her-2, which is present in about 30% of women diagnosed with breast cancer. Her-2 is a cell surface receptor that leads to unregulated cell growth. Women having tumors with the Her-2 marker experience a poorer clinical outcome. One possible approach to treatment is to use antibodies to Her-2 to directly target breast cancer cells. Daryl Drummond, Ph.D., is attaching Her-2 antibodies to liposome particles to deliver chemotherapy drugs directly to the interior of breast cancer cells. In contrast, Malcolm Mitchell, M.D., is trying to find portions of the Her-2 protein that will stimulate a cytotoxic T lymphocyte recognition of breast cancer cells. The idea is to generate a cell-mediated immune response, rather than just antibodies. Joseph Lustgarten, Ph.D., will attach an immune-enhancing cytokine to Her-2 antibodies to ‘mark’ tumor cells for attack by the immune system. From a totally different perspective, Gordon Louie, Ph.D., is using the diphtheria toxin x-ray crystal structure as a way to model a novel molecular interaction with a natural ligand for the Her oncogene family, called heregulin.
Several new innovative treatment awards are strongly in the clinical realm. Surgeons need more advanced molecular markers to use in diagnosing breast biopsies. Shanaz Dairkee, Ph.D., will study the molecular pathology of breast samples to see if defects are present even when the visual histological appearance is normal. There is evidence to suggest that the stage of the menstrual cycle can effect the outcome of breast cancer surgery. Hillary Klonoff-Cohen, Ph.D., Helena Chang, M.D., Ph.D., and Hungyi Shau, Ph.D., will approach this issue by examining the differential effect of surgery during different phases of the menstrual cycle on disease outcome. The key strength here is collaborative association of oncologists and epidemiologists brought together in the Translational Research Collaboration (TRC) funding mechanism. Physicians and surgeons are confused and frustrated that, while so many breast cancer ‘markers’ are known, we are lacking in follow-up studies to see how effective they are in predicting clinical outcome and survival. Shelley Enger, Ph.D., Michael Press, M.D., Ph.D., and Jon Greif, Ph.D., are assembling a team (TRC Pilot) and developing a strategy for collecting and evaluating information on Her-2, p53 (a tumor suppressor gene) and Bax (which regulates apoptosis, or normal cell death) from a sample of patients, and analyzing this information with respect to the therapy provided and subsequent survival status. Finally, we know that only a portion of women actually respond to any given therapy, and this is most frustrating for those women considering chemotherapy. Silvia Formenti, M.D., Peter Danenberg, Ph.D., and Franco Muggia, M.D., will be studying whether they can identify tumor markers that will predict the response of a tumor to the drug, Paclitaxel, with or without radiation before actual treatment begins.
