Innovative Treatments and Models of Care

Previously, in the second cycle of funding, the Breast Cancer Research Council decided that there is a need to foster, (i) both late preclinical studies (those that could move rapidly to the clinical phase) and early clinical studies of innovative treatment modalities, and (ii) novel/non-traditional models of care specifically targeting patients with breast cancer. The Innovative Treatment Modalities and Models of Care priority areas were continued in this third granting cycle. Through these priority areas, the primary charge to the BCRP researchers is to expeditiously bring treatments from the laboratory to the clinic. The studies funded in the section are geared to delivering the maximum impact to breast cancer treatment in California. Nineteen new grants were awarded for these two priority areas in this funding cycle.

Three models of care awards were funded. One project will study the impact of bringing a comprehensive breast cancer center to ‘underserved communities’ on the access to medical care and the non-adherence to medical recommendations (Jay Harness, PI). Two other awards are based on ideas generated from community groups to assist in support of underserved women with breast cancer and designed by partnerships between these groups and experienced research scientists. In one of these studies, an intervention to assist rural women diagnosed with breast cancer is being designed and tested as a substitute for support groups, which are not feasible in many rural areas (Mary Anne Kreshka and Cheryl Koopman, co-PIs) . In the other study, the psychosocial effects of a retreat for low-income women with breast cancer, who also are often not able to attend regular support groups, will be measured (Shoshana Levenberg and Ellen Levine, co-PIs).

Sixteen newly funded grants focus on emerging treatment modalities. These are studies that investigate questions in breast cancer treatment that are especially innovative and have a high potential payoff. Eleven projects are for comprehensive, detailed translational research efforts in established laboratories. Next, three of the funded grants are two-year postdoctoral fellowships, which were designed to bring young investigators into the field of breast cancer treatment. Lastly, two Translational Research Collaboration (TRC) pilot awards were funded. The BCRP developed the TRC award in order to encourage intensive, multidisciplinary communication between basic science researchers and breast cancer clinicians, thereby moving treatments from the laboratory to the clinic as quickly as is feasible. We anticipate that the TRC pilot projects will provide the preliminary research and collaboration interactions necessary for an application for a full, three-year TRC in the next funding cycle.

One TRC pilot award will allow the researchers to build the infrastructure to study the effectiveness of factors that inhibit blood vessel formation (angiogenesis) in reducing tumor growth and metastasis, and the diagnostic utility of vascular endothelial growth factor in detecting breast cancer (Marc Shuman, Randall Hawkins, Laura Esserman, co-PIs). A second pilot award will allow the researchers to collect the preliminary data towards identifying the biomarkers that predict which breast tumors will respond to the relatively new chemotherapeutic drug, Paclitaxel (Silvia Formenti, Peter Danenberg and Franco Muggia, co-PIs).

The treatment modality research grant and fellowship awards primarily use the methods of immunology, endocrinology and molecular biology to find new ways to treat breast cancer. The use of the patient’s immune system to battle the disease using a previously under-investigated component of the immune system, dendritic cells, is featured in one grant (Jeffrey Weber, PI), and bioengineered immune receptors to better target and kill tumor cells are the focus of two other grants (Sherie Morrison, PI and Jeffrey Smith, PI). The development of more efficient inhibitors of enzymes that make estrogen (Masato Tanabe, PI), the inhibition of the production (Joel Gottesfeld, PI) and the function (Yoko Fujita-Yamaguchi, PI) of factors that stimulate tumor growth are three endocrinologically-based therapeutic tactics funded this cycle. Two other grants look at ways to treat tumors by blocking factors inside the cell that cause tumors to grow, thereby improving the effectiveness of chemotherapy (Yuefeng Lu, PI and Dan Mercola, PI). One grant is utilizing a gene therapy delivery system that should inhibit the formation of blood vessels that support tumor growth (Robert Debs, PI). Four other studies design new drugs or employ molecular-based strategies to interfere with the ability of the breast cancer cells to attach to the molecules in immediate contact (extracellular matrix) with the tumor (Nurulain Zaveri, Renata Pasqualini, Joseph Konopelski and Qing Zhou, PIs). These projects could also offer the potential of inhibiting angiogenesis. Finally, one fellowship grant investigates the mechanism of bone loss that is associated with metastatic breast cancer (Herve LeCalvez, PI). These projects should open up exciting new areas of breast cancer treatment.

Translational Research Collaboration (TRC) Awards


Biochemical Correlates for Clinical Response to Paclitaxel

Silvia Formenti, M.D.
Peter Danenberg, Ph.D.
Franco Muggia, M.D.

University of Southern California

Since only chemotherapy (and hormonal therapy in specific subsets of patients) can reduce the risk for breast cancer to relapse and to affect survival, the recommendation for most breast cancer patients is to undergo adjuvant chemotherapy (after surgical removal of the tumor). Unfortunately, most patients derive no benefit from adjuvant chemotherapy. Only about one in ten women who are treated by conventional adjuvant chemotherapy benefit from it while the remaining nine derive no benefit, either because they would have fared as well in the absence of systemic treatment or because they will recur despite it. Patients and doctors share the frustration of lacking ways to pre-select that one patient in every ten who would benefit from adjuvant chemotherapy.

The goal of this pilot study is to identify those patients who are most likely to benefit from treatment with paclitaxel. We think that it is very useful to monitor the effect of chemotherapy on the original tumor instead of waiting until the primary cancer has been removed and then giving chemotherapy. We will test the effect of a dose-intensive regimen of paclitaxel, a very active drug in breast cancer, on the primary tumor. We will monitor the tumor for 6 weeks of treatment then we remove the area of the breast that contained the original tumor (by lumpectomy or mastectomy, per patient’s choice). By measuring the extent of residual cancer, we predict that we will be able to find a large range of tumor response: in some women the cancer cells may have disappeared while in others the tumor may persist. By assuring that some of the original tumor tissue is collected through a small biopsy before paclitaxel treatment, we can study the original biological features of each tumor and attempt to correlate them with the response we found in the surgical specimen after paclitaxel therapy. This method may enable us to identify specific markers in the original tumor that can predict for the clinical response to paclitaxel.

We plan to accrue a total of 45 women over 18-24 months to this study, by joining the efforts of three separate institutions, the University of Southern California, Mayo Clinic-Jacksonville and New York University. By collaborating we will be able to generate very important preliminary data in a relatively short time. Our pilot study is unique in having selected a paradigm of clinical research that by using the pre-operative dose-intensive, single agent paclitaxel we will be able to: a) measure chemosensitivity directly in the breast cancer and b) understand which molecular characteristics in the tumor may predict for response.

The work proposed may generate the basis for designing more patient-specific chemotherapy treatment, to optimize the chances of success of the available chemotherapy agents. In the future, studies like this will enable us to match the optimal treatment to each breast tumor, based on the tumor characteristics. We expect a more patient-specific use of chemotherapy agents to impact favorably on the mortality from breast cancer.

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Inhibition of Angiogenesis in Breast Cancer

Marc Shuman, M.D.
Randall Hawkins, M.D., Ph.D.
Laura Esserman, M.D.

University of California, San Francisco

New blood vessel growth, termed angiogenesis, is necessary for breast tumors to grow and spread (metastasis). The focus of our research is a human protein called Vascular Endothelial Growth Factor (VEGF), which appears to be required for angiogenesis in many types of tumors, including breast cancer. Over 50% of breast tumors appear to secrete VEGF, where it acts in the immediate environment to attract blood vessel growth. In animal studies, an antibody directed against VEGF was found to inhibit tumor growth and metastases, and this therapy had no apparent toxicity. In addition, the presence of VEGF in breast tumors could be utilized as a diagnostic marker, and as a measure of potential aggressive metastasis to other sites in the body. Further, in the treatment of breast cancer by current and future therapies, VEGF detection modalities could be used as both a measure of and an endpoint of a successful clinical outcome.

This pilot study would lay the groundwork for examining the effectiveness and diagnostic utility of an antibody against VEGF in the treatment of women with advanced breast cancer. A key to this effort will be employing a ‘humanized’ VEGF antibody. These particular antibodies, unlike antibodies produced in animals, have the advantage of not triggering a patient’s immune response which quickly removes the antibody from the body. Thus, the treatment and diagnostic uses could extend for sufficient time to arrest and reverse tumor growth. In related work, we hope to develop new methods of radiographic imaging using both ‘positron emission tomography’ (PET) and ‘Magnetic Resonance Imaging’ (MRI). These detection methods allow us to non-invasively assess angiogenesis in breast tumors and the effectiveness of anti-VEGF therapy. In addition, we would be able to predict from PET and MRI pre-screening, those women with breast cancer who are best suited for anti-VEGF treatment. Our parallel research goal is also to investigate the basic biology of angiogenesis specific to breast cancer. We anticipate identifying other factors that promote tumor blood vessel growth. Thus, new targets will be generated to treat those women whose tumors do not show VEGF production, but with active angiogenesis and a high risk of metastasis.

The impact of developing a therapy that is relatively non-toxic and effective in both the adjuvant and metastatic treatment settings would have a significant impact on the economic and human cost of breast cancer in California.

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ITaMoCA Awards

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Gene Therapy for Breast Cancer

Robert Debs, M.D.
California Pacific Medical Center

Great progress has been made in the last 10 years in identifying and understanding the causes of breast and other human cancers. Tumor development, progression, and spread are accompanied by a wide spectrum of known genetic mutations, deletions, or abnormal gene expression events. Thus, it is becoming increasingly clear that specific pieces of human DNA, or genes, ultimately produce breast and other cancers in humans. Yet our ability to directly neutralize or correct these genetic abnormalities is poorly developed. Our interest is to explore methods to deliver specific, corrective genes directly to the cancerous tumors themselves. The application of this approach has been elusive for a variety of reasons including, (i) stability and delivery from the blood, (ii) uptake and incorporation by the cancer cells, and (iii) inadequate production of the desired gene product (i.e., protein) by the cells. Despite these difficulties, the long-term outlook for gene therapy in cancer remains promising. Already, work performed in our laboratory on the delivery of a normal copy of the gene which, when abnormal causes cystic fibrosis, has lead to a human trial.

In order to fix the defective genes in breast cancer patients, it is necessary to deliver the therapeutic DNA molecules or genes directly to the tumors. Our laboratory uses microscopic fat globules, called liposomes, to transport the corrective DNA to the tumor site. We have already shown that we can efficiently and safely deliver a number of genes to cancerous tumors in mice, which reduce the growth and metastasis of melanoma. We will now extend our method for DNA delivery to tackle breast cancer treatment in experimental animals. The primary breast cancer function we will address is angiogenesis, the growth of new blood vessels allowing both tumor growth and spread. We will incorporate an anti-angiogenic gene, angiostatin, in a liposome delivery system, and follow up by measuring such parameters as tumor growth, vascularity and metastasis. In parallel studies we will deliver the tumor suppressor, p53, and the immune activator, GM-CSF, to determine their potential as anti-tumor gene therapy agents.

Pending the successful application of our methodology in animals, we will attempt to direct this approach towards the treatment of human patients with breast cancer. We envision a future breast cancer treatment scenario where the specific abnormalities of individual patients are selectively addressed by a gene therapy approach.

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Receptor Antibody-Enhanced Chemotherapy for Breast Cancer

Yoko Fujita-Yamaguchi, Ph.D.
Beckman Research Institute of the City of Hope

We now know that cancer arises as a result of a series of molecular alterations in normal cells including overexpression of growth factors that could facilitate uncontrolled cell growth. Insulin-like growth factor (IGF)-I and -II are growth factors. IGF-I is thought to play an important role in development after birth with the majority of circulating IGF-I coming from the liver. IGF-II, on the other hand, is assumed to be an important growth factor in fetal and new born development. Abnormal overexpression of these IGFs in cancer has increasingly been recognized. It is known that IGF-I and -II are potent growth factors for a variety of cancer cells, including breast cancer cells. In addition, several lines of experimental evidence suggest that an increase in IGF-II expression is associated with malignant progression of breast cancer cells whereas a decrease of IGF-II expression results in growth inhibition of these cells. The growth effects of IGF-II and -I are mediated through the IGF-I receptor (IGFIR), that is, IGFs tell the cells to start growth by binding to IGFIR.

Based on these observations, we have designed innovative experiments that interrupt IGFs/IGFIR growth signaling by introducing a "receptor antibody" that keeps IGFs from binding to IGFIR. Treatment of breast cancer cells with existing IGFIR-specific antibody (aIR-3) has been shown to reduce IGF-stimulated cell growth. Our anti-IGFIR antibody, IH7, is apparently even better at interrupting IGF/IGFIR signaling. This fact indicates that IH7 is a desirable candidate for testing the effect of receptor antibodies on cancer cell growth inhibition. Two versions of IH7 antibodies have been produced using "state of the art" bioengineering technologies. One form should be able to inhibit IGF actions from outside cells whereas the other should be able to inhibit IGFIR function inside cells. In addition, we plan to test this receptor antibody therapy in conjuction with chemotherapy for breast cancer in order to maximize its potential effect. We expect that the proposed "Receptor antibody-enhanced chemotherapy" would not only enhance sensitivity of cells with lots of IGFIR to chemotherapy, but also affect other cancer cells that don’t have as much IGFIR. It is hoped that the proposed study will lead to the development of new therapeutics for breast cancer treatment. The enhancement strategies for chemotherapy by anti-growth factor receptor antibodies are now believed to lead to new strategies for clinical interventions shown to inhibit IGF-stimulated cell growth.

Antibody strategies to inhibit IGF-stimulated breast cancer cell growth have never been tested. Accomplishment of such antibody strategies would lay the foundation for the development of future breast cancer therapeutics utilizing chemo/immuno/gene therapy. Thus, this proposal will fit into one of the Priority Issues included in Cycle III, "Innovative Treatment Modalities".

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Inhibitors of the Breast Cancer Her-2/neuGene

Joel Gottesfeld, Ph.D.
The Scripps Research Institute

This research plan involves the development of new drugs for the treatment of human breast cancer. It is well established that human breast cancer is associated with uncontrolled cell growth due to the expression of certain cancer genes. One such gene, called Her-2/neu, is mutated and/or over-expressed in 25-30% of breast tumors. This oncogene makes the cells more responsive to growth factors leading to uncontrolled cell growth. Her-2/neu is present on the cell surface, and drug development is underway, including use of specific antibodies, to block its function. The inherent potential limitations with this approach encouraged us to develop alternate approaches to ‘manage’ Her-2/neu expressing breast cancers.

Our research plan involves the design and study of small molecules to target and inactivate Her-2/neu. The target in our approach is not the Her-2/neu cell-surface receptor, but rather the gene encoding Her-2/neu in the cell nucleus. Every gene is associated with DNA segments that regulate its expression into RNA/protein. We will ‘attack’ the DNA regulatory part of the Her-2/neu gene by use of novel inhibitory molecules, called ‘pyrrole-imidazole polyamides’. These small molecule inhibitors bind to their gene targets with very high affinity and selectivity. Thus, ideally the inhibition is only at the desired target gene. The net effect is to ‘clog’ the normal mechanism of gene usage by the cell, thus ‘turning-off’ a specific gene. The targeted cell is not killed, but genetically reverted to a normal state. The obstacles to overcome are (i) the specific structure of the inhibitory molecule, (ii) selection of the Her-2/neu target DNA sequence to achieve selectivity, and (iii) determination of the optimal dose to reduce potential toxicity. As a test of the effectiveness of our approach, we will determine whether these compounds will inhibit both the expression of genes that are regulated by estrogen and the expression of the Her-2/neu oncogene itself in human breast cancer cells.

The identification of such small molecule inhibitors of specific gene expression has the potential to generate a new class of drugs for the treatment of human breast cancer.

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The Breast Care Center: Innovative Care for The Underserved

Jay Kenneth Harness, M.D.
Northern California Cancer Center

Minority, low-income, and medically underserved women are at higher risk of dying from breast cancer than women from less disadvantaged groups. These adverse differences in survival rates have been linked in part to delays by some women in these groups in obtaining care and to not following through with medical recommendations. Delays and missed treatments have been linked in turn to institutional, economic, social, cultural, and linguistic barriers that can impede these patients’ access to care and limit the ability of providers to deliver quality services in a way that effectively addresses the patients’ multiplicity of needs.

We propose to conduct a comparative evaluation of an innovative organizational model - the Breast Care Center (BCC) - for improving the delivery of breast cancer control services to underserved women. A BCC is a "one stop" center that provides all of the outpatient services essential to breast cancer care and organizes them to ensure maximum efficiency and timeliness. It brings together the full range of cancer-related medical specialties and social services, and supports and tracks patients through all phases of cancer control: patient education; screening; timely resolution or diagnosis; prompt initiation of treatment; long-term follow-up; and patient support services. Although this model’s effectiveness has been established in university and private hospitals, it has rarely been used, and never adequately evaluated, in facilities providing care to underserved patients.

This study is a collaboration among four public hospitals in San Francisco Bay Area counties. Two of them utilize the BCC model and two, which will serve as control sites, do not. We will examine whether county hospitals providing care to breast cancer patients through the BCC model, compared to county hospitals providing traditionally organized care, offer more comprehensive and coordinated services; provide more appropriate and timely care; track patient progress better; produce greater patient satisfaction with care; and achieve better patient adherence to treatment recommendations. Our aims are to: (1) document how the BCC concept is implemented in two county hospitals, to assess the extent to which BCC goals are being met, and to identify institutional barriers to meeting these goals; (2) test the hypothesis cited above; and (3) use study data to develop a comprehensive BCC model and evaluation procedures for the four-county area and other areas serving multicultural underserved women.

We will identify points in the health care continuum at which institutional barriers impede patients’ access to quality care, and examine the impact of the BCC model on alleviating those barriers. We will also establish a comprehensive system for collecting and managing data on breast cancer in the four-county study area to facilitate this and future research.

We expect that underserved patients using the BCC will find it easier to access needed services, obtain quality care, and adhere to medical recommendations. By promoting early detection and facilitating diagnosis, treatment, and follow-up, the model has a strong potential to address barriers to successful outcomes and improve survival rates for the most vulnerable segments of our population.

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Novel Drugs to Inhibit Breast Cancer Metastasis

Joseph Konopelski, Ph.D.
University of California, Santa Cruz

The change from a normal to a cancerous cell is accompanied by changes in the biological molecules at the surface of, and surrounding that cell. Some of these new biomolecules are unique enough to allow their use for diagnostic and treatment purposes. One protein component of the matrix of molecules that both surrounds a cell and forms the basement membrane is called laminin. Breast cancer cells attach to laminin using a specific laminin-binding protein (LBP), a cell surface protein. LBP expression provides an excellent prognostic indicator in breast cancer, and shows a positive correlation with progression in many solid tumors. This correlation likely results from the ability of LBP to enhance tumor cell attachment with the basement membrane as a prerequisite to spread in the body, or metastasis. Although laminin is a large protein, only a small part, called peptide 11, is involved in cell binding. When injected into test animals, peptide 11 can effectively block tumor spread to the lung and tumor angiogenesis (blood vessel growth). Unfortunately, other properties of peptide 11 make it unsuitable for immediate therapeutic applications. Thus, we have initiated a multi-disciplinary collaboration focused on structural analysis of peptide 11 to develop novel drugs for inhibition of breast cancer invasion and metastasis.

Our project pursues a chemical approach to prepare, structurally analyze, and test laminin peptide 11-derived unusual amino acids and related analogs (peptidomimetics). The desired molecular properties include (i) increased hydrolytic stability, (ii) protein inhibition properties, and/or (iii) unique conformational restrictions. These properties are measured by both enhanced protein and cell binding and drug stability when administered by animal injection. Peptide 11 is very attractive as a lead, ‘parent’ compound for developing a novel therapeutic agent. We expect that molecules resembling peptide 11 will not have additional, unwanted biological effects. Thus, our research goal is to block movement of breast cancer cells to spread through the body. Our present inability to address tumor cell spread, or metastasis, is a challenge to develop novel drug treatments based on our current understanding of the molecular processes involved. Finally, our research has added potential application of being able to detect a protein (LBP) present on breast cancer cells, thus offering both a method of early detection and identifying those tumors with a dangerous metastatic potential.

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Enhancing Breast Cancer Sensitivity to Chemotherapy

Daniel Mercola, M.D., Ph. D.
Sidney Kimmel Cancer Center

Current therapies and technologies to treat and detect breast cancer are not optimal. Tumor cells have elaborate mechanisms to evade drug therapy, which is especially true of chemotherapy. A more effective approach to chemotherapy would be first to render the cancer cells more sensitive. Thus, the chemotherapeutic dose could be lowered to reduce the side effects. A critical problem posed by cancer cells is their ability to selectively ‘activate’ certain genes causing unregulated cell growth. Our work focuses on a group of genes called the "immediately early genes", which play a fundamental role in the control of cell growth. One of these early genes is called c-jun. The c-jun protein acts as a regulator of gene expression, one aspect of which is to enhance DNA repair mechanisms. We have discovered that this effect is greatly increased by Jun kinase, an enzyme that phosphorylates c-jun, thereby enhancing its gene stimulating role. Thus, Jun kinase is essential in allowing breast cancer cells to escape the killing effect of certain chemotherapeutic drugs, such as the DNA-damaging agent cisplatin.

In one phase of our research, we genetically altered various human cancer cells, including breast carcinoma cells, to produce an unphosphorylatable (inactive form) of c-jun. This c-jun competes with the normal c-jun, which effectively shuts off this pathway inside the cell. Thus, these cells grow much more slowly and fail to generate tumors in test animals. Significantly, these c-jun-defective cancer cells were sensitive to the chemotherapeutic agent, cisplatin. Our current project is to develop a new method to block c-jun in cancer cells that has potential therapeutic application. This method is called ‘antisense’. We will work with a leading pharmaceutical company in ‘antisense’ techniques, ISIS Pharmaceuticals Inc., to pursue a novel approach to block c-jun. To date, lead ‘antisense’ compounds have been developed, which work in the laboratory to block the synthesis of Jun kinase and inhibit cell growth. We are now ready to try this in human breast cancer cells to determine whether Jun kinase-targeted ‘antisense’ will both block DNA repair and sensitize these cells to killing by cisplatin. Next, we will apply the ‘antisense’ approach to treat human breast cancers grown in test animals.

The strength of our approach rests in our ability to tackle both excess growth of tumor cells and their resistance to chemotherapy. ‘Antisense’ technology to battle breast cancer has not been fully exploited, since new cancer-specific targets constantly become available and the methodology itself is under-appreciated as an ‘adjunct’ therapy.

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ITaMoCA Awards 2

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Antibody Fusion Proteins for the Therapy of Breast Cancer

Sherie L. Morrison, Ph.D.
University of California, Los Angeles

The management of residual disease is a central problem in breast cancer. Despite extensive efforts, relapse remains a critical and generally fatal problem in high-risk breast cancer patients. Conventional treatments such as chemotherapy are limited in their efficacy by various toxicities and by tumor resistance. Additional modalities that will achieve better results are needed. A variety of investigators have suggested transferring genes for molecules that stimulate the immune system into tumor cells to augment the immune response against them. Many of these strategies involve manipulation of tumor cells outside the patient, which is technically difficult to implement and does not target all tumor deposits. We propose an alternative approach in which molecules that stimulate the immune system can be selectively delivered to the tumor cells of a metastatic/residual nodule by a tumor specific antibody. It is anticipated that the molecules will engender a potent immune response against the targeted tumor.

Using genetic engineering techniques, we have joined antibodies to molecules that are stimulators of the immune system producing "antibody fusion proteins". Over the last several years work in our laboratory has focused on producing antibody fusion proteins with novel biologic activities. We now propose to further explore the use of genetically engineered antibody fusion proteins as therapeutic agents specifically attempting to augment and potentiate the patient’s immune system against breast cancer. We will use antibodies specific for the breast cancer associated molecules her2/neu and CEA. Overexpression of her2/neu has been associated with poor prognosis making it especially promising for targeting residual disease in breast cancer. To these antibodies we will join either IL-2 or B7-1, both of which have been shown to be effective in increasing the immune response against tumors. We anticipate that the antibody will carry the immunostimulatory molecule to the site of the tumor where it will stimulate the human immune system to recognize and destroy the cancer cells, which otherwise might escape conventional treatment.

The first generation of these fusion proteins has been made. The fusion proteins are of the expected size and carry out antibody related activities as well as the activity of the immuno-stimulator molecule that was joined to the antibody. The next step will be determining the properties of the fusion proteins in animals and their effectiveness in eliciting an anti-tumor response. We will test the effectiveness of the fusion proteins in eliminating established breast cancer using tumor models in mice. We have introduced the genes for her2/neu and CEA into mouse tumors so that we can investigate the efficacy of our treatment in immunocompetent mice. Using different tumor models, we and others have shown that antibody fusion proteins can have a superior protective effect due to specific stimulation of the immune response. We are now in an excellent position to determine if this approach can be extended to the treatment of minimal residual disease in breast cancer.

The present studies represent a novel extension of previous reports and address the priority issue "Innovative Treatment Modalities" that can contribute significantly to reducing the cost of breast cancer in California.

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Superfibronectin: A Novel Anti-Breast Cancer Agent

Renata Pasqualini, Ph.D.
The Burnham Institute

The spread of breast cancer to other, distant organs is a process known as metastasis. The most common organs affected by metastasis are bones, lungs, and liver. Metastasis makes breast cancer almost incurable. Early detection and treatment addresses cancer spread indirectly, but no present treatment directly blocks metastasis. In order to spread, the cancer cells break free from the primary tumor, enter the lymph or blood, and leave the circulation at preferred organ sites. This final phase of circulation arrest and escape involves the binding of tumor cell surface receptors to protein components that surround cells, the extracellular matrix (ECM). One component of the ECM is fibrobnectin (FN), a complex protein also present in the blood. Thus, the tumor cells in transit in the blood could be ‘attacked’ and neutralized by therapeutic agents that target their receptors for FN.

Our approach is to study modified forms of FN to disrupt the process of breast cancer cell adhesion specific for metastasis. We have recently developed a polymer of FN, called superfibronectin (sFN), which can block cell adhesion and migration in the laboratory. Therefore, we reasoned that sFN could act on cancer cells in transit, either in the circulation or about to metastasize, by coating them and preventing their adhesion to distant target organs. In this project, we will expand our studies on sFN for prevention of metastasis in mice, using human breast cancer cells. In preliminary experiments, sFN had a strong anti-metastatic activity. Initial studies also showed that sFN can restrict, in addition to metastasis, the growth of primary breast cancer by mechanisms not yet fully understood. Thus, a second goal of our work will be to follow-up on the mechanism of the tumor growth-inhibiting properties of sFN. In contrast to other common cancer treatments, such as chemotherapy or radiotherapy, no toxic side effects were observed in the treated animals.

Thus, the treatment effects observed so far and the absence of toxicity makes sFN an outstanding candidate to treat breast cancer, especially since metastasis is not targeted by the available treatments. Our eventual goal is to establish a breast cancer model for future clinical trials using sFN. An unexpected finding of our preliminary work indicates that metastasis may be integrated with tumor growth. Thus, metastasis-targeted approaches could also combat either the primary tumor or the cells already having spread to provide an additional benefit.

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Targeting T Cells to Breast Cancer

Jeffrey W. Smith, Ph.D.
The Burnham Institute

The premise of this grant is that recombinant DNA technology can be used to create human immune cells that will target and kill breast cancer. The primary function of the human immune system is to fight infection brought about by viruses and bacteria. The immune system will not normally recognize or attack one’s own tissues. Because cancer is really derived from one’s own tissues, the immune system has difficulty in identifying cancerous cells and often fails in combating this disease.

We have developed a new protein engineering technique called "protein loop grafting", which allows us to rapidly and precisely change the function of a protein. We plan to use this strategy to alter the function of proteins in the immune system called T-cell receptors. T-cell receptors can actually direct the immune system to attack specific targets. Our strategy is to alter the T-cell receptor so that it can bind to multiple proteins on the surface of breast cancer cells. We will accomplish this by using protein loop grafting. We hypothesize that the immune cells that contain the engineered T-cell receptor will display "targeted immunity". In other words, they will seek out and kill breast cancer cells. If the studies we propose here are successful, they will solve a major problem in cancer and immunity because we will be able to redirect the immune system so that it can recognize and destroy tumors. Once these concepts have been tested in the laboratory, a gene therapy approach can be developed to alter a patient’s own lymphocytes so that they can bind to and kill breast cancer cells. The potential rewards of this ITaMoCA are: the development of an entirely novel way of manipulating the binding function of the T-cell receptors; the creation of the first T-cell receptor with the ability to recognize two separate proteins on the surface of breast cancer cells, an advance that is likely to assist in applying immunotherapy in a tumor-selective manner; and a potential immunotherapy for patients with advanced stage breast carcinoma.

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Novel Enzyme Inhibitors for Estrogen-Dependent Breast Cancer

Masato Tanabe, Ph.D.
SRI International

This research project involves advancement of a truly novel approach to treatment of estrogen-dependent (estrogen-receptor-positive, ER+) breast cancer: an orally active, potent drug that blocks estrogen formation in breast tumor cells by inhibiting one of the enzymes that make estrogen but does not display any undesired estrogenic side effects. SRI International has already performed extensive preliminary studies to design and develop this new class of drug candidates, estrone sulfatase inhibitors (ESIs), and screen promising candidates for estrogenic activity and inhibitory activity in cell culture. Our preliminary studies have identified five lead candidates that hold great promise for treatment of ER+ breast cancer.

ESIs block the formation of estrogens, which have been shown to support the growth of breast tumors in women. Estrogens in breast tumors originate either by uptake of estrogens circulating in the blood or by local formation from other hormones. In postmenopausal women, in whom breast cancer is most common, breast tumor levels of estrogens—particularly the hormone estradiol—are considerably higher than the levels in the blood. There are two enzyme systems that produce estrogen in tumor cells: aromatases and sulfatases. Estrone sulfatase is responsible for producing the major circulating estrogen in the blood, estrone sulfate, which locally provides breast tumor cells with their main source of estradiol. Estradiol is widely thought to be responsible for development of breast carcinoma. This sulfatase pathway produces 10 times more estrogen than the aromatase pathway (the second highest producer of estrogen in breast tumors), and estrone sulfatase activity levels are 1000 times higher than aromatase activity levels in breast tumors.

Use of aromatase inhibitors to deprive tumor cells of estrogen is currently an important method of endocrine treatment for breast cancer, but 90% of the estrogen in breast tumors appears to originate by way of the sulfatase pathway. It is not surprising that our new ESIs appear to block estrogen formation better than the aromatase inhibitors, and they may be very effective either alone or in combination with aromatase inhibitors.

The goal of the research is to perform chemistry and biology studies that will identify the most promising of our five lead ESIs and provide a solid basis for advancing the new agent toward clinical use. We propose a 2-year program that will provide this basis and lay the foundation for attracting a pharmaceutical company to help complete the development of the new agent and make it available for treating patients with estrogen-dependent breast cancer.

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Peptide-Pulsed Dendritic Cell Therapy for Breast Cancer

Jeffrey S. Weber, M.D., Ph.D.
University of Southern California

In this project, we intend to test whether normal cells from the blood stream of breast cancer patients called dendritic cells have the ability to stimulate immunity against breast cancer. The dendritic cells are normally present in small numbers in the blood, and stimulate immunity very strongly against viruses, bacteria, cancer cells and other foreign invaders. Scientists have developed a way to grow these cells in large numbers outside the body within a week after removing a sample of blood and exposing the white blood cells to growth factors that encourage the growth of dendritic cells. In this research project, dendritic cells will then be exposed to a synthetic piece of a substance present on breast cancer cells called "CEA" that is present on tumors from two thirds of patients with breast cancer. Dendritic cells will be grown for a week outside the body and then exposed to a fragment of the "CEA", followed by infusion in large numbers into the veins of patients with breast cancer that has spread widely and cannot be cured with chemotherapy, radiation or surgery. Increasing doses of the cells will be given to groups of at least three patients with breast cancer in order to find out whether the dendritic cells cause side effects when given twice over a period of two weeks. Stimulation of the immune system against breast cancer will be measured before and after the dendritic cells are given by removing a sample of blood for analysis in the laboratory and by other tests. Shrinkage of tumor will also be measured. In this 2 year trial of 24 patients with metastatic breast cancer, we will ask whether patients that receive CEA dendritic cells can increase their immunity against breast cancer without side effects, and whether increased immunity against breast cancer can cause shrinkage of tumors. The long term goal of this initial study is to find ways to educate the immune system to recognize and destroy breast cancer cells even though the presence of the tumor and the chemotherapy agents used to shrink the breast cancer have caused decreased levels of immunity. The results of this initial CEA dendritic cell study will determine the course of future studies depending on whether shrinkage of tumor is seen, in which case a follow-up study will be performed to confirm that CEA dendritic cells are effective for the treatment of metastatic breast cancer. If no tumor shrinkage is seen, but immunity against breast cancer is boosted, then patients will be treated with CEA dendritic cells and then have a blood sample removed to try to grow another type of white blood cell called killer T cells that can be directed against breast cancer. This use of dendritic cells to stimulate immunity against breast cancer is supported by work showing that in the mouse and in humans, dendritic cells are the strongest stimulators of the immune system. This immunologic approach to breast cancer is an attempt to harness the body’s natural defenses to eliminate tumors by boosting the immune response directed against a substance found on cancer cells.

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Blocking Stromelysin-3 to Inhibit Breast Cancer Metastasis

Nurulain Zaveri, Ph.D.
SRI International

Metastasis, the spread of cells from the primary cancer to distant sites in the body, is the most fearsome aspect of breast cancer. Despite significant advances in breast cancer detection and treatment, most deaths from breast cancer are due to metastasis that is resistant to conventional therapies. Occult or hidden micro-metastases may already exist in women diagnosed with tumors. Metastasis consists of a series of linked, sequential steps that enable tumor cells to secrete tissue-destroying enzymes, which allow cells to break away from the original cancer. Therefore, current drugs that only stop cancer growth may not always halt the metastatic spread of the cancer. Recent studies on breast cancer biopsies reported high levels of a new enzyme, stromelysin-3 (ST-3), particularly from patients who later developed metastasis. ST-3 was found to be involved in the early steps of metastasis, which convert a tumor cell to a metastatic cell. ST-3 belongs to a well-known class of zinc-containing enzymes (matrix metalloproteinases, MMPs), but it has several unique properties. Interestingly, it does not directly break down the extracellular matrix surrounding tumor cells, but instead appears to bind to and inactivate a natural inhibitor of other tissue-destroying enzymes. This then allows these other enzymes to degrade the matrix, leading to metastasis.

The goal of this proposed research is to target the unique aspects of ST-3 to design and develop a drug that will specifically block the action of ST-3 and thereby halt the process of metastasis in its early stages. We will use a multidisciplinary approach for drug design, which consists of organic synthesis, computer modeling and biological testing. We will target the active site of ST-3 by using small protein-based (dipeptide) inhibitors that incorporate a strong zinc-binding component. We will evaluate the effectiveness of these inhibitors by using both purified ST-3 and whole cell systems to better represent a natural, biological model. Feedback from this biological evaluation will be integrated into the drug design to obtain a therapeutically viable anti-metastatic compound.

Novel antimetastatic drugs would be especially valuable in combination with surgery and/or chemotherapy. Current widespread use of screening mammography has increased the detection of very small, early-stage breast cancers. Thus, drugs that prevent metastasis would serve to combat recurrence and assure a complete and lasting cure.

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Posdoctoral Fellowship Awards

The Role of Meltrin-a in Breast Cancer-Associated Bone Loss

Herve LeCalvez, Ph.D.
The Burnham Institute

In advanced stages of breast cancer, there is spread to and involvement of the bone. This results in bone loss and an excess of circulating calcium (humoral hypercalcemia). Both bone loss and hypercalcemia are the result of physiological destruction of the bone by resident cells called osteoclasts, which are stimulated by breast carcinomas. New osteoclasts are formed as a result of cell fusion from previously mononuclear, precursor cells. This fusion event is regulated by a cell surface protein, meltrin-a, which may serve to attach two cells together in the initial phase of osteoclast formation. An additional component of this process is believed to be an integrin receptor, likely avb3, which in other situations allows cells to associate with the extracellular matrix. Given this level of information, there is obviously a lack of critical molecular information with which to develop a therapeutic approach to inhibit bone loss in breast cancer. In addition, our work could find application in another critical disease of importance to women, osteoporosis.

We propose to first investigate the specific regions within the extracellular domains of the meltrin-a receptor. This involves two approaches: (i) introducing parts of the meltrin-a into cells that resemble osteoclasts to better identify their function, and (ii) preparing specific antibodies to parts of meltrin-a and using the antibodies to block functions of this protein. Our hypothesis is that meltrin-a has two functional domains, one for triggering the fusion of the plasma membranes of two adjacent precursor cells (cell fusion event), and another for an earlier phase of cell-cell interaction via the integrin, avb3. Thus, the second aim of our work is to identify the cell surface receptor of the meltrin-a, and then to describe their interaction. Our hope is that we can develop information regarding the interaction of the meltrin-a with its receptor for the purpose of developing an inhibitor having therapeutic applications. Our initial thought is that this new inhibitor could be peptide-based, if this receptor is related to the integrins.

It is anticipated that the results from our study will provide the following advances relevant to bone wasting associated with breast cancer: (i) new basic science information on the critical molecular mechanism for the sequential steps in osteoclast precursor fusion, and (ii) initiate the process of identifying new lead compounds and applications to reduce bone loss.

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Modulation of Drug Resistance by Protein Kinase Inhibitors

Yuefeng Lu, Ph.D.
Stanford University

Chemotherapy, in association with surgery and radiation therapy, is very important for the treatment of breast cancer. In chemotherapeutically drug-sensitive breast cancer cells, the drug molecules are able to enter and accumulate inside the cell. Upon reaching a certain concentration, the drugs cause cellular damage that usually turns on the intrinsic pathways that lead to cell death, a process called apoptosis or programmed cell death. Unfortunately, the effectiveness of chemotherapy can be diminished by the development of drug resistance. Drug resistance can occur via two important mechanisms: by decreased drug accumulation inside the cells (or increased drug exclusion), and by the failure of the drugs to induce the programmed cell death. Increased drug exclusion has been correlated to the presence of higher amounts of a protein called P-glycoprotein, or P-gp, on the cell surface. This protein works like a pump that excludes drugs from cells and prevents drug accumulation inside. Programmed cell death is a highly regulated cellular event. Drug-resistant cells may have defects in the regulation of this pathway or they may have mutations in one or more of its key components. Consequently, even when drug accumulation inside the cell is high, the cell may still be able to survive.

A protein that has been shown to play important roles in both drug exclusion and programmed cell death is Protein kinase C, or PKC. PKC adds phosphorous groups onto other proteins, which usually results in dramatic changes in the activities of these proteins. PKC has more than twelve different forms, called isozymes, at least six of which are present in breast cancer cells. Though they have similar structures and requirements for activation, each isozyme may regulate different cellular events, or have different or even reverse effects on the same event. Drug resistant breast cancer cells have an elevated PKC activity, however the role(s) of individual PKC isozymes in drug resistance are largely unknown.

This laboratory has developed several isozyme-selective PKC inhibitors. Application of these inhibitors, which are not available in other laboratories, would allow us to examine the impact of each inhibitor on drug exclusion and the programmed cell death, and therefore the function of individual PKC isozymes in drug resistance. PKC isozymes that promote drug exclusion and/or suppress drug-induced cancer cell death would be identified. Isozyme-selective inhibitors of these PKC isozymes may represent leading compounds for the development of novel agents that circumvent drug-resistance and potentiate the effects of breast cancer chemotherapy. Drugs developed based on these inhibitors should have fewer side effects and greater effectiveness because they are isozyme-selective.

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Breast Cancer Gene Therapy Using a Metastasis Inhibitor

Qing Zhou, M.D., Ph.D.
University of Southern California

At the time of diagnosis, over 60% of breast cancer patients will have disease that has spread (metastasis) from the primary site in the breast to other parts of the body. While the primary tumor can be removed, there is no adequate therapy for preventing the spread of the tumor to secondary sites. We have been studying an anti-metastatic protein from the venom of the Southern copperhead snake, called contortrostatin (CN). This protein blocks the function of a group of cell surface receptors called integrins, which are the key cellular receptors that allow cancer cell attachment, movement, and migration in the body. Thus, the integrins on cancer cells are prime targets to develop new drugs and treatment modalities. Presently we use an experimental model where mice are implanted with human breast cancer cells in the mammary fat tissue to test CN for blockage of tumor growth and metastasis. Daily injections of CN into these tumors slows their growth rate and also reduces their metastatic spread by >95%. We have evidence that this effect of CN is due to a combination of three effects which include (i) impeding invasion of the cancer cells into blood vessels, (ii) preventing the attachment of cancer cells to the blood vessel wall, and (iii) blocking new blood vessel growth (angiogenesis) into tumors.

Gene therapy is one of the most promising recent developments in medicine. Using a non-disease causing retrovirus, new genes can be integrated into the chromosomes of cells. These genes can make new proteins with therapeutic functions. We plan to use this approach to introduce the CN gene into cells called myoblasts, which are precursors of muscle cells. The myoblast cells will be implanted into the tumors, or other appropriate sites in the animals, to produce CN. We anticipate that the CN so produced will significantly inhibit the growth and metastasis of the breast cancer.

Our project uniquely combines gene therapy with a potential anti-metastatic agent, which will result in continuous delivery of CN directly to the site of tumor growth. This research may lead to a clinical application addressing an important need for anti-metastatic therapy in breast cancer patients.

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CIRC Awards

A Community-based Workbook for Helping Rural Cancer Patients

Mary Anne Kreshka, M.A.
Sierra Nevada Memorial Cancer Center
Cheryl Koopman, Ph.D.

Stanford University School of Medicine

Women diagnosed with breast cancer deserve immediate and continuing education and emotional support. When a woman is newly diagnosed with breast cancer, she is faced with extremely difficult decisions about her treatment. Often she must make these decisions quickly, without knowing much about the long term effects of these choices, and while trying to cope with the fact that she has a life-threatening illness. When a woman is nearing the end of her treatment for breast cancer, she must face the prospect of living day-to-day with the uncertainty of whether her cancer will return.

In many communities, support groups have been developed by breast cancer survivors or cancer treatment centers to address these needs. In rural counties, however, many women who would like to participate in support groups live too far away or do not have reliable transportation. To respond to this need La Loba, a grass roots breast cancer support group in Nevada County, plans to create a user-friendly workbookjournal that provides facts, figures and personal experiences by other women who have been diagnosed with breast cancer. This workbookjournal, entitled "One in Eight", will include such topics as how to relate to doctors and medical technicians. how to talk to family and friends, how to cope with hair loss, energy loss, and other side-effects of chemotherapy. It will include humor and space for personal reflection. Information about local and regional resources, such as books, organizations and public agencies, will help direct women in their search for education about breast cancer and its treatment. The theme of the book will be to encourage and support women taking charge of their lives, being informed of their choices, and participating actively in their treatment.

Women from this local support group have teamed up with researchers from the Stanford University School of Medicine to form the Sierra-Stanford Partnership. This partnership, between rural breast cancer survivors, rural care providers and researchers specializing in supportive interventions for cancer patients, plans to develop and test the helpfulness of "One in Eight" by offering it to breast cancer patients in Nevada, Sierra, Amador, Placer, and Eldorado Counties in the Sierra Foothills. The rural community partners will write the workbook and will interview patients to receive feedback about the workbook. The research team will determine whether "One in Eight" helps women to better cope with and adjust emotionally to their cancer, and to make better breast cancer treatment choices.

The hope of the Sierra-Stanford Partnership is to reduce the human and economic costs of breast cancer by reaching rural women who do not have access to current forms of education or support, and help them make the best possible breast cancer treatment choices.

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The Efficacy of A Retreat for Low-Income Women with Breast Cancer

Shoshana Levenberg, B.S.N.
Charlotte Maxwell Complementary Clinic
Ellen Levine, M.P.H., Ph.D.

California Pacific Medical Center Research Institute

The Charlotte Maxwell Clinic is a non-profit organization that offers complementary care, such as massage and acupuncture. to low income women with cancer. The Clinic does not treat cancer but rather offers relief from the side effects of cancer and its treatments -- nausea, pain and fatigue. What happens in the Clinic’s treatment rooms is magical: being touched and cared for with kindness, competence and respect. What happens in the waiting lounge is at least as important: the transformative power of women connecting, telling their stories. sharing their laughter and pain. The Clinic has an active census of 60 low income women, 40 of whom have breast cancer. At least 25 of those women are eager to plan and participate in a 3-day retreat offering information, resources and coping strategies for living with breast cancer.

The idea of a retreat to assist women living with chronic disease is based largely upon the success of WORLD, a grassroots organization by and for women with HIV, which has held ten 3-day retreats in the last 5 years. Our retreat will be planned by a committee of clients and staff from the clinic, all of whom are living with cancer. That every planner has personally faced the day-to-day challenges of cancer is the critical element in ensuring a genuine sense of community ownership from the beginning. The retreat itself will have numerous workshops such as "Partnering with your Doctor", "Treatment Options", and benefits counseling, all to be identified by the planning committee. In addition to sharing information, it will be a time for women to connect, in small and large groups, to share both fears and hopes in a safe place. We see the retreat as simply one tool for breaking the life-draining isolation of living with cancer. It is not an end unto itself and for many women it will be a beginning. We hope to build on this beginning by studying the effectiveness of the retreat. We will ask the women who attend to evaluate how helpful the retreat was for them.

We will also contact the women one month after the retreat and ask them if they have been able to obtain needed resources. We hope to use the results of this project to obtain additional funding in order to expand our study of the benefits of such community-based interventions.

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