Innovative Treatment Modalities & Early Detection: Searching for and Improving Chances for the Cure

Translational Research Collaboration- Full Research Awards
Innovative, Exploratory and Developmental Awards – Type I
Innovative, Exploratory and Developmental Awards – Type II
Postdoctoral Fellowship Awards
Training Program Award

The BCRP has a desire to move basic science projects into the pre-clinical transition stage for new therapeutic development to treat and prevent breast cancer. The Innovative Treatments priority issue offers the opportunity for researchers, both clinical and basic science, to be funded for innovative research to explore translational goals. Thus, these research projects often represent a critical 'bridge' from the bench to the bedside.

The BCRP funded 11 new grants for innovative treatments of breast cancer in Cycle V. These projects cover a wide range of topics from uses of Chinese herbs to the emerging technology of 'gene chips' for the diagnosis of breast cancer. With the explosion of new genetic information many of these projects work at the 'gene level'. This is a tremendous improvement over the previous simple endpoints of growth, metastasis, and cell death for therapeutic evaluation. Thus, as more genetic information on breast cancer becomes available these findings will fill in the genetic puzzle of the disease. It is clear that there will not be one, single effective therapeutic or preventative strategy for all breast cancer(s). Treatments of the 21st century will recognize the diversity of the disease and approach it from many opportunistic angles. Both patients and physicians will be offered many choices that combine the need to eliminate the cancer and to maintain the quality of life.

The BCRP priority issue of Earlier Detection is related to innovative treatments, since many of the projects involve the discovery of biomarkers of the disease, as well as projects focused on medical physics and topics related to early detection technology.

The BCRP funded five projects in Cycle V on earlier detection. Carolyn Kimme-Smith will direct a medical physics training program that exposes students to pathology, laboratory, and clinical issues relevant to breast cancer. The remaining four grants relate to biomarkers of breast cancer. This topic overlaps with innovative treatments, because it is possible that such markers could serve both a detection and prognostic function, as well as play a role in decisions on therapeutic intervention. First, Jeffrey Smith will explore a novel molecular approach to detect breast tumor proteases that might be released into the circulation. Secondly, H. Phillip Koeffler is funded to use a yeast-based system to detect proteins secreted from breast cancer cells to discover potential biomarkers for future study. Third, Dave Hoon will investigate the potential of sampling blood to isolate circulating tumor-specific DNA to test for markers associated with the disease. This approach has the potential to substitute for surgical biopsy in order to gain critical clinical information regarding tumor status and possible treatment strategies. Finally, Robert Cardiff will study biomarkers in mouse systems to gain information on the relationship of current and potential markers to stage and predict the progression of breast cancer.

The discovery of new drug compounds, refining their key structural elements and mode of action, and exploring the appropriate clinical uses continue to be major areas of investigation. Traditional medicines are recognized as potentially safe and effective treatment methods. Unfortunately, Western medicine has not yet been able tap this reservoir of new drug options. Perhaps as many as 50% of breast cancer patients and survivors use herbs, green tea, and other plant compounds to self-medicate as an adjunct therapy. Debasish Tripathy is funded to study traditional Chinese medicine by investigating botanical used in Chinese medicine for anti-tumor activity. The aim is to gain more information on which agents are most effective as a first-step for additional clinical studies. Michelle Tabb is investigating a novel intracellular protein that could provide a key common link between the action of phytoestrogens (i.e., plant estrogens), the drug tamoxifen, and Vitamin A in regulating breast cancer cell growth. This project could clear up confusion about why seemingly unrelated compounds act to control cancer. Two newly-funded projects investigate compounds derived from cruciferous vegetables (e.g., Brussels sprouts) for their effect on breast cancer. Gary Firestone is continuing a project initially supported by the BCRP in 1997 to investigate indole-3-carbinol (I3C). This compound will inhibit breast cancer cell growth and it appears to work effectively in estrogen-independent tumors. He plans to develop derivatives of I3C to overcome hurdles of potency and bioavailability in tumor models representative of the human disease. Working with Dr. Firestone is Liqun Zhang, who is funded to examine how I3C works at the molecular level in breast cancer cells. It appears that I3C inhibits the passage of cells through checkpoints necessary for cell division and growth. Finally, Mai Nguyen is funded to purify and study novel compounds derived from a Chinese palm tree for their activity in inhibiting the process of blood vessel growth, which is necessary for metastasis and tumor growth.

Two newly-funded projects focus on detecting and evaluating biomarkers for breast cancer that are associated with clinical diagnosis and treatment, rather than earlier detection. First, Stefanie Jeffrey will utilize gene chips to survey breast cancer samples for gene expression patterns. These gene chips have thousands of human gene sequences, and they can simultaneously detect increases and decreases in gene expression in a rapid, automated fashion. Dr. Jeffrey's interest is whether this technology can be applied to smaller biopsy samples from patients at a point where therapy and treatment decisions are critical to a successful outcome. Secondly, Shelley Enger and Michael Press (co-PIs) are funded for a 3-year Translational Research Collaboration to study the relationship of existing biomarkers, such as Her-2 and p53, in predicting the success of different adjuvant therapies from large numbers of women diagnosed and treated for breast cancer in the Kaiser Permanente and University of Southern California systems. It is critical to know how women respond to chemo-, radio-, and hormonal treatments following their diagnosis. Women who have breast cancer with the Her-2 growth receptor oncogene have new therapies (i.e., the Herceptin monoclonal antibody) available. But, research continues on Her-2 to better target and treat breast cancer. Michael Campbell is exploring a molecular approach to combine parts of the Her-2 protein with a virus in an attempt to create a novel immunovaccine. Richard Pietras will study how radiation therapy can be combined with Herceptin treatment, and how this combined treatment works on the intracellular signaling pathways inside of breast cancer cells.

The BCRP funded two research projects that explore the use of radiotherapy to target and treat breast cancer. First, Xiaofei Wang is examining how radiation-induced DNA damage leads to DNA repair. If these intracellular signaling proteins could be rendered inactive, then breast cancers could be made much more radiosensitive prior to therapy. Finally, Michelle Winthrop is funded to target radionuclides (i.e., radioactive molecules, rather than externally applied iodizing radiation) to breast cancer. Her approach is to make bispecific antibodies that both will transport the radionuclide and will selectively bind to cancer when injected into the circulation.

top

Translational Research Collaboration- Full Research Awards


Can Molecular Markers Predict Response to Adjuvant Therapy?

Shelley Enger, Ph.D.1 and Michael Press M.D., Ph.D.2
1Kaiser Permanente Southern California and 2University of Southern California

We will conduct a study to determine definitively whether examining specific characteristics of a tumor can help identify patients most likely to benefit from chemotherapy and other breast cancer treatments. When planning a patient's treatment, the physician has detailed information regarding how advanced the disease is, but the physician has very little information available to determine how likely a patient is to respond to a given treatment. An important exception is the role of estrogen receptor status in planning for endocrine therapies (such as tamoxifen), for which earlier research demonstrated that the benefit was restricted to women whose tumors were positive for estrogen receptors. Since then, interest has been mounting to reveal other tumor characteristics that may predict response to therapy.

In this study, we will examine the ability of specific genetic markers to predict how well patients will respond to therapy. To achieve this goal, we will collect detailed tumor and treatment information from the medical records of 1,695 female Kaiser Permanente members who were diagosed with invasive breast cancer from the late 1980s to the middle 1990s at the Kaiser Permanente Medical Center in San Diego, California. Tumor blocks for each of these patients will be examined for changes in the HER2/neu, p53, and BCL2 genes. Using this information, we will determine how often these changes occur in breast tumors and we will determine whether a patient's response to specific treatments depends on the presence of these genetic changes. If the results of this research suggest a change in the traditional methods of planning patients' treatment strategies, this information will be made available to clinicians through the investigators' publications and presentation of the results in cancer journals and at scientific meetings. In addition, we will have the unique opportunity of establishing a cohort of breast cancer patients to examine possible long-term consequences of these gene changes.

This project will also support the development of innovative and sound scientific hypotheses resulting from the collaborative partnership between Kaiser Permanente Southern California, one of the largest providers of health care in the state of California, and the University of Southern California, a premier cancer research institution. One immediate benefit of this collaboration is access to a large breast cancer patient population with tissue blocks available for nearly all patients. Thus, we have an important opportunity to advance knowledge in this field, and answer important questions regarding the clinical management of breast cancer.

The ultimate goal of this study is to provide physicians with tools that will allow them to better tailor breast cancer treatment regimens to the individual patient, reducing morbidity associated with the therapies and improving survival among breast cancer patients.

top

Innovative, Developmental and Exploratory Awards – Type I


Identification of Novel Secreted Proteins of Breast Cancer

Phillip H. Koeffler, M.D.
Cedars-Sinai Medical Center

Successful treatment of breast cancer still depends on the early detection of the disease. Mammography and MRI depend on physical detection and have limitations as to cost, availability of experts to interpret the images, and access to women in rural and underserved areas. What is needed is a simpler, informative, and quantitative method with acceptable risks. Our interest is the development of serum markers for breast cancer, which could greatly facilitate discovery of disease recurrence and progress in treatment regimens. Currently, the CA15-3 serum marker is used for monitoring therapy of breast cancer, but it is fraught with lack of specificity and sensitivity, rendering it of little use as a screening tool.

Our aim is to develop novel serum biomarkers for breast cancer. For this project we will use a molecular technique that is designed specifically to detect proteins secreted from cells and possibly present in the blood of women having breast cancer. We are using a new yeast-based molecular screen that detects the genes for proteins that are secreted from cells. This employs a 'signal sequence trap' technique, because secretory breast cancer proteins are expected to contain a molecular 'signal' for cellular export. Initially, we have constructed a cDNA library that has been enriched for breast cancer-related genes. This library has been placed in our yeast-based signal sequence trap vector in order to identify genes encoding secreted proteins. The first part of the project will be to identify breast cancer cDNA clones of interest. Then, we will confirm these clones, express them as protein fragments, and prepare specific antibodies. We hope to develop this information in future work to utilize these antibodies against breast cancer secreted proteins as potential cancer detection agents.

We hope that this technology will evolve to the point where both known and novel breast cancer secreted proteins can be measured in a manner similar to the PSA test for prostate cancer diagnosis. Moreover, a by-product of this project may be the discovery of novel secreted or membrane-associated proteins specific to breast cancer, such as growth factors or their receptors, which may contribute to clarifying the pathogenesis of these cancers.

Novel Breast Cancer Anti-Angiogenic Compounds

Mai Nguyen, M.D.
University of California, Los Angeles

Two decades of experimental evidence have demonstrated that the growth and metastasis of breast cancer are dependent on angiogenesis, the development of new blood vessels. Although over 30 anti-angiogenesis compounds are in clinical trials, we are focusing our search on natural compounds that could readily be given as safe, oral inhibitors. Our search has revealed that an extract from the plant Livistona, a palm tree, can inhibit experimental angiogenesis and suppress breast tumor growth in animal model systems. Preliminary analysis showed that the active compounds are stable to heat, acid, and most likely are small organic molecules. So far, there has been no observed toxicity in mouse experiments.

In this study, we propose to further isolate and identify the exact chemical structure of the active anti-angiogenesis compound(s). This purification/chemical analysis will be accomplished by ion exchange chromatography, gas chromatography coupled with mass spectrometric analyses, nuclear magnetic resonance, infra-red spectroscopy, and X-ray crystallography. The resulting purified compound(s) will be tested for anti-angiogenesis activity in cellular assays and in mouse tumor models.

The current list of drugs in development for treating angiogenesis looks promising. However, plant-based compounds have the potential advantage of use in more preventive (chronic) settings and in an orally active form. We think that the discovery of a potent anti-angiogenic drug with low toxicity would potentially be very useful in the treatment and prevention of breast cancer, especially in high-risk women or survivors at risk for recurrence.

top

Innovative, Developmental and Exploratory Awards – Type II

top


HER2/Neu DNA Vaccines for Breast Cancer

Michael J. Campbell, Ph.D.
University of California, San Francisco

New and innovative treatment strategies, such as immunotherapies, are clearly needed to improve outcomes in breast cancer, which too frequently recurs or progresses despite aggressive multimodality therapy. Cancer vaccines have the potential to both treat existing cancer and prevent its recurrence. In addition, breast cancer vaccines may be an ideal intervention for preventing ductal carcinoma in situ (DCIS), a very early form of breast cancer, from progressing to invasive cancer.

We are developing and evaluating vaccines targeted against the HER2/neu protein. This protein is present on the cell surface of over 50% of DCIS tumors and 30% of invasive breast cancers. The HER2/neu protein acts as an "antenna" on the surface of the cancer cell, receiving signals that cause these cells to grow out of control. In previous work we have explored immunotherapy approaches in mice, which have been altered to express HER2/neu and spontaneously develop breast tumors. When these mice were vaccinated with a HER2/neu protein vaccine, only half of these mice developed breast cancer compared to 90% of control animals. Thus, the potential is evident, but detailed improvements are needed at this stage of our therapeutic development.

We now propose a major refinement in our vaccine strategy, which will use novel alphavirus-based HER2/neu vaccines. This approach is a special type of immuno-gene therapy in which segments of the HER2/neu gene are introduced into alphaviral DNA expression vectors. These vectors will be introduced into host cells as plasmid DNA. Once inside the host cells, they will exploit viral mechanisms for amplification to yield high level expression of the HER2/neu. This produces an antigen against which the host can generate an immune response. These vectors have been designed such that infectious particles cannot be created. Thus, they are a safe system for vaccine development. As an additional aim, we plan to introduce immunomodulatory proteins (e.g., GM-CSF and IL-2) in the alpa viral vectors to determine if these will enhance the anti-HER2/neu immune response. These experiments will utilize mouse models that endogenously express HER2/neu. In addition to the effect of the DNA vaccine on tumors we will measure parameters of the immune response, such as the relative role of CD4+ and CD8+ T-cells, T-cell immunity transfer, and cytokine assays.

Results from this project will lay the foundation for developing and producing novel human HER2/neu vaccines for breast cancer.

back  top

Oncogenes, Breast Cancer Progression and Biomarkers

Robert D. Cardiff, M.D., Ph.D.
University of California, Davis

Proteins and other chemicals that can "mark" cells in cancers are used in immunohistochemistry (IHC) both to determine whether a women's breast cancer is "good" or "bad" ("prognostic" indicators indicating whether the likely outcome of treatment and subsequent course of the disease will be favorable or unfavorable) and to develop treatment strategies for the cancer. However, the stains that "mark" the cells are variable from patient to patient. Some physicians feel that they cannot be used to predict the outcome for the individual patient. As a result, some HMOs will not pay for the entire panel of tests, and prognostic IHC is not the "standard of practice" in all hospitals. Why are these results so variable?

Our hypothesis is that the cancer causing genes (oncogenes) that initiates the cancer control the patterns of transition of normal breast tissue to atypical, to pre-malignant, to malignant and to metastatic breast cancer. These patterns are non-random and highly predictable. A corollary hypothesis is that each oncogene causes unique predictable patterns of protein expression (appearance) that can be detected as biomarkers. The interactions of the cancer causing oncogenes and these factors (biomarkers) modify the appearance of the tumor and the processes of angiogenesis (vessel formation), invasion and metastasis. These processes determine how the cancer spreads.

This hypothesis implies that the pattern of expression of a set of biomarkers depends on the gene, or genes, that initiate(s) the cancer. The hypothesis can be tested by immunohistochemical analysis of a panel of normal, precancerous and malignant breast tissues with known activated oncogenes. Genetically engineered mice are a ready source of breast tissues with such defined oncogenes. They also have demonstrable histological transitions between normal, precancerous and malignant breast tissue. The breast tissues from these defined-oncogene mice will be used to test the hypothesis using a panel of antibodies against established and candidate breast cancer biomarkers.

The Specific Aims of this project are to use immunohistochemistry to:

  1. Characterize the distribution of breast cancer biomarkers in a panel of transgenic mouse mammary tissues including normal, premalignant, malignant and metastatic.
  2. Determine whether the distribution of breast cancer biomarkers is altered by additional transgenes (bi- or tri-genic mice) or knock outs of suppressor genes.
  3. Test candidate breast cancer biomarkers in the same system.
 

These studies will utilize a collection and expertise that is not available anywhere else in the world. They should resolve the controversy surrounding the use of biomarkers, making them much more reliable predictors of the cancer outcome and provide a rational basis for therapy.

back  top

Indole Derivatives as Novel Breast Cancer Therapeutics

Gary L. Firestone, Ph.D.
University of California, Berkeley

A critical problem in treating breast cancer is the lack of effective therapeutics directed against estrogen-independent forms of the disease, which respond less favorably to the anti-estrogens (e.g., tamoxifen). In addition, there has been less research aimed at the identification of active ingredients and molecular mechanism of action for dietary components in breast cancer prevention and treatment.

Our interest is focused on a compound called indole-3-carbinol (I3C), which is found in Brassica-type vegetables (e.g., cabbage, broccoli, and Brussels sprouts). When ingested, dietary I3C has been shown to be converted into several natural acid-catalyzed products that have multiple anti-tumor bioactivities. First, using both estrogen responsive and nonresponsive human breast tumor cell lines, we have discovered that I3C activates a novel anti-proliferative pathway that is independent of the estrogen receptor. The net effect is to inhibit the cell cycle, through a loss of cell expression of CDK6 (cyclin-dependent kinase 6) and a reduction in activity of another cell cycle protein, CDK2. This pathway prevents the breast cancer cell from dividing by blocking its passage through the cell cycle. Secondly, we have found that a combination of I3C and tamoxifen will synergistically block the growth of estrogen-responsive breast cancer cells.

The next step is to refine our understanding of how I3C works in the various hormone states in breast cancer, and to approach this problem with the aim of developing these natural compounds into chemotherapeutic and chemopreventative drugs. The specific aims of this project are to (1) generate and characterize new classes of structurally related I3C with improved potency; (2) identify the I3C derivatives that more effectively induce a cell cycle arrest, inhibit CDK6 gene expression and CDK2 activity, especially in estrogen-independent cancer; and (3) study our novel compounds in cellular and animal models of growth and tumor formation. Our project is unique in that it combines laboratory research on breast tumor biology and mechanisms with drug development and toxicology expertise of our collaborators.

Much interest is currently given to the use of the anti-estrogen, tamoxifen, as a chemopreventative agent for high-risk groups of women. However, this approach is severely limited in potential by the issues of both acquired drug resistance and the fact that a substantial percentage (up to 40%) of breast cancers are estrogen-independent in nature. Given the limited information about the role of indole-type compounds produced in vegetables in the treatment breast cancer, our study will provide the necessary first experimental information needed for the development of novel chemotherapeutic, or perhaps chemopreventative, strategies that effectively utilize potent derivatives of I3C.

back  top

Clinical Utility of Breast Cancer DNA Markers in Plasma

Dave S.B. Hoon, Ph.D.
John Wayne Cancer Institute

It has become more apparent that in cancer patients, identification of genetic changes in tumors can be highly informative in assessing the outcome of treatments. Multiple genetic changes are required for tumors to start growing, continuing growing and become more aggressive. Although multiple genetic markers in breast cancer have been defined, studies of how they can be used to diagnose breast cancer and predict disease progression are still limited. However, genetic changes (DNA markers) in breast cancer should prove to be valuable as prognostic tools. Traditionally genetic markers are tested only in tumors that have been surgically removed, which limits the assessment of the disease progression to the genetic status of the tumor when it was removed. We have developed an innovative molecular diagnostic assay to assess multiple genetic markers in a small amount of blood (less than 5 ml) from breast cancer patients. This assay provides a unique approach in monitoring genetic changes occurring during tumor progression without the need to obtain tumor tissue through invasive surgical procedures. The assay is highly informative, more tumor-specific than conventional assays, provides an inexpensive means of rapid testing, and offers an improved method for diagnosis and prognosis of breast cancer disease. By correlating these genetic analyses with clinico -pathological factors, the assay provides an exciting new approach for assessing breast cancer progression.

The diagnostic significance of genetic markers for tumor suppressor genes (loss of heterozygosity) in plasma will be assessed in patients undergoing sentinel lymphadenectomy. Sentinel lymphadenectomy removes the first node (sentinel node) to receive metastases from the primary breast tumor. This innovative surgical procedure, which accurately stages disease progression in patients, detects small instances of tumor spreading (micrometastasis), and reduces the side effects associated with standard lymphadenectomy, was pioneered by JWCI and is now used throughout the world. The genetic assay will be conducted in conjunction with the sentinel lymphadenectomy procedure to improve efficacy in diagnosis and prognosis. This provides a highly innovative approach for detecting and understanding disease spreading.

The successful completion of these studies will hopefully provide an assay for diagnosis and prognosis as well as evaluation of therapeutic interventions. To date, the decision about a treatment regimen has been predominantly based on the physical, biochemical and genetic characteristics of the tumor. This assay will provide a new way to use genetic markers as measures of disease progression and potentially improve our ability to tailor the most effective treatment(s) to individual breast cancer patients, thus improving the management of breast cancer disease in California. This study endeavors to achieve this goal by incorporating and combining the fields of surgery, pathology, molecular genetics, medical and radiation oncology, and nursing.

back  top

Breast Cancer Gene Expression Using Amplified Core Biopsies

Stefanie S. Jeffrey, M.D.
Stanford University School of Medicine

When normal breast epithelial cells turn into breast cancer there are profound changes in the pattern of genetic expression. These changes include both gain and loss of gene function, as well as changes in the level of expression for critical genes. This 'pattern' of gene expression in cancer opens the door to improved diagnosis and treatment based on a 'molecular diagnosis'. The molecular diagnosis of cancer interfaces with emerging technologies in genomic research and this has the potential to improve medical decision making at earlier stages of the disease.

Our research project utilizes a newly-developed, state-of-the-art gene microarray or "gene chip." This consists of a glass microscope slide containing over 9000 gene spots in a square area less than 0.75 inches per side. In order to survey the expression level of breast cancer genes, the RNA is extracted from a tumor sample and converted (reverse transcribed) into cDNA. This cDNA needs to be representative for diversity, quality, and quantity compared to the starting tissue sample. These cDNA samples are placed on the microarray and binding occurs between the cDNA and the individual gene 'spots' on the chip. These represent 'matches' and provides a readout for the relative expression level for over 9000 genes, many whose function is currently unknown. Based on the use of color dyes and special scanners, we can quickly determine specific patterns of gene expression in a given breast tumor sample.

Unfortunately, the current use of the 'gene chip' technology is limited to large tumors (>1.5 cm), because of the need to isolate substantial quantities of mRNA. This project seeks to improve the utility of this procedure for smaller breast tumor samples obtained from routine core biopsies. In this project we will test RNA 'scale-up' amplification from small breast samples using an antisense RNA amplification technique. We will be able to compare core biopsies from large tumors with the traditional RNA preparation method using the same tumor sample. In addition, we can perform multiple core biopsies from a single tumor to see if there are consistent gene expression patterns as a measure of tumor heterogeneity.

Development of this gene survey approach will put more diagnostic power in the hands of surgeons and oncologists to understand the molecular features of breast tumors. For example, the expression levels of genes characteristic of angiogenesis, metastasis, or aggressive growth (e.g., HER2/neu) would indicate the need for individual types of therapy. This is potentially a vast improvement over pathological appearance and today's limited biomarker technology. Thus, we eventually hope to use this amazing technology to help women with small tumors in order to predict whether or not they need chemotherapy, and if they do, which drugs would work most effectively. Ultimately, specific and currently unrecognized gene products may be identified for the development of novel targeted cytotoxic therapies.

back  top

New Radiation Therapy for HER-2-overexpressing Breast Cancer

Richard Pietras, M.D., Ph.D.
University of California, Los Angeles

Radiation therapy is an important component in breast cancer management in most patients. Patients treated with breast conservation surgery routinely receive additional therapy with external beam radiation. After total mastectomy with dissection of the lymph nodes, radiation therapy is also recommended for women with large primary tumors, extension of tumor beyond the lymph nodes, or the occurrence of tumor in four or more lymph nodes. The other important role of radiation therapy is in the palliation of symptoms caused by either the primary tumor or metastatic spread of the tumor to distant sites, such as bone. Radiation therapy generally causes injury to the DNA of breast cancer cells. In most cases, this radiation-induced DNA damage is irreversible, and the irradiated cancer cells are killed. However, in other cases, the cancer cells manage to repair the radiation-induced damage to their DNA, and the malignant cells then continue to grow and spread. The latter condition leads to higher chances for relapse of the breast cancer in the affected patient. In fact, some recent clinical studies show that the risk for breast cancer recurrence after treatment with surgery and radiation is higher among breast cancer patients with tumors demonstrating an abundance of a particular marker protein, termed HER-2. This protein is present at the blood surface of the cancer cell and acts as an antenna to attract growth-promoting signals from outside the cell. Abnormally high levels of the HER-2 growth-promoting protein can be found in 25-30% of human breast cancers. A new FDA-approved treatment for the management of patients with metastatic breast cancer is an antibody directed against this HER-2 protein. This new biologic agent, called Herceptin, can be used alone or in combination with certain chemotherapy drugs. Recent studies from our laboratory suggest that therapy of HER-2- overexpressing breast cancers with Herceptin enhances tumor sensitivity to radiation. In addition, combined therapy with Herceptin and radiation causes tumor remissions in breast cancers that failed treatment with radiation alone. This benefit appears to be specific to cells with large amounts of HER-2 and does not occur in cells with normal amounts. A clinical advantage may be achieved in combined Herceptin-radiation therapy if Herceptin selectively radiosensitizes tumor cells or if the two agents act additively and selectively. The possibility that radiation and Herceptin could act selectively against certain breast tumor cells is the subject of the present proposal.

We propose to further evaluate Herceptin-induced radiosensitivity and plan to conduct extensive preclinical studies to evaluate the safety and efficacy of this new therapy. In addition, we hope to learn more about molecular events associated with radiation-antibody treatment in order to further improve utili-zation of this innovative approach. Human cancer cells damaged by radiation may be especially vulnerable to injury if they are also deprived of essential growth-promoting signals provided by the HER-2 growth -promoting protein. We plan to exploit this weakness in HER-2- overexpressing breast cancers in order to develop a new approach to radiation therapy with better clinical outcome and quality of life for affected breast cancer patients in California and elsewhere.

This project proposes an innovative treatment modality that has not been used before. The research team is multidisciplinary, with primary contributions from clinical investigators in Intenal Medicine and Medical Oncology and Radiation Oncology. The impact of this preclinical trial is truly translational. The proposed research exploits a postulated DNA repair-regulatory circuit involving cell surface HER-2 proteins and is intended to lead directly to clinical trials of radiation therapy in combination with Herceptin in patients with HER-2- overexpressing breast cancers.

back  top

Protease Fingerprinting to Diagnose Breast Cancer

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

There is still a great need to develop more convenient, cost effective, and accurate methods of detecting and tracking breast cancer. The availability of simple and rapid blood tests to screen for breast cancer could vastly increase the number women who are routinely screened. A blood test that detects breast cancer could, like cholesterol screening and PSA for prostate cancer, potentially be used to detect disease stage, changes in disease progression, and to monitor therapy. It would be particularly important for high-risk women, breast cancer survivors at risk for recurrence, and younger women who are difficult to screen accurately using mammography. The two hurdles that must be overcome, 1) the test must recognize many parameters, and 2) the test must be highly sensitive.

This study proposes a method for identifying the "fingerprint" of breast cancer. We plan to study changes in a group of proteins called proteases, which cut other proteins into smaller segments causing them to break down. This tumor-associated proteolytic activity causes the localized changes in the surrounding extracellular matrix, which are key to blood vessel formation (angiogenesis), tumor cell migration, and spread to other parts of the body (metastasis). Our hypothesis is that some of these tumor proteases are released into the blood, and are potentially detectable using emerging technologies. Thus, we will test blood samples from tumor-bearing animals for their ability to cleave peptides that are known substrates for cell proteases. In another phase of the project we will confirm that proteases of interest are associated with the tumor and not originating from other sources. A protease 'fingerprint' would be diagnostic for the clinical parameters associated with breast cancer.

In this phase of the project, we hope to identify 50+ unique proteases indicating the presence of breast cancer. We also suspect that another 50+ will indicate that a tumor has metastasized, or is capable of metastasizing. We anticipate that the results of this study will point the way for a future test of this approach in human breast cancer patients.

back  top

Laboratory Testing of Chinese Herbs Used for Breast Cancer

Debasish Tripathy, M.D.
University of California, San Francisco; Mt. Zion Breast Care Center

Traditional Chinese Medicine (TCM) is a treatment modality that has been used for centuries to cure cancer, prolong life, increase the quality of life for cancer patients, and more recently to ameliorate the side effects of Western therapies. The lack of evidence-based clinical trials coupled with the proliferation of anecdotal and case report studies of TCM make it an important treatment modality for further study. TCM use in the Bay Area is widespread and frequently used by our patient population, thus it can be studied using standard research models at our institution. There is a high degree of conformity among TCM practitioners in terms of both diagnoses and treatments due to the extensive historically-based documentation of TCM treatment modalities. We propose to obtain laboratory data on herbs commonly used for breast cancer in order to prioritize herbal formulae for future Phase I/II clinical trials.

We plan to perform laboratory analysis of a number of botanical agents that have been traditionally prescribed by TCM practitioners for metastatic breast cancer (MBC) patients. We have pilot data on a few herbal compounds that show anti-tumor activity, and we will use a similar expanded methodology to screen approximately 50 more botanical agents. We will test for the ability of these compounds to cause breast cancer cells to divide using an MTT assay, to commit suicide (apoptosis) using APO-BRDU and to grow using clonogenic assays. The compounds will be tested for their effects using both pulsed and continuous exposure. For herbal extracts with activity, the responsible components will be identified using salt exchange column and high performance liquid chromatography fractions. Promising herbs or combinations could then be entered into Phase I/II clinical trials using tumor response endpoints.

back  top


Postdoctoral Fellowship Awards


SXR: A Novel Target for Breast Cancer Therapeutics

Michelle M. Tabb, Ph.D.
University of California, Irvine

The steroid hormone estrogen has been shown to be involved in the development and growth of many breast cancers. Accordingly, drugs that block the effects of estrogen, such as tamoxifen, are helpful in the treatment and risk reduction of breast cancers. Other compounds such as plant-derived estrogens called phytoestrogens have also demonstrated effectiveness in preventing breast cancer. Phytoestrogens are mimics of the hormone estrogen, and are found abundantly in soy-based foods as well as in small amounts in whole grains, seeds, fruits and vegetables. The anti-breast cancer potential of these estrogen mimics is suggested by the lower incidence of breast cancers in women of Asian populations that consume larger levels of these compounds compared to women in Western populations that consume much lower amounts. In addition, other compounds such as anandamide and Vitamin A derivatives that are seemingly unrelated to each other or to tamoxifen and phytoestrogens also have potential as effective anti-breast cancer agents. Because these compounds are all seemingly unrelated, it has been difficult to put all of them into a common pathway that can explain their anti-breast cancer effects.

Research carried out under this proposal will investigate a common mechanism by which each of these diverse compounds is active against breast cancer. The potential mechanism involves a protein in the cell called SXR that has demonstrated interaction with tamoxifen, phytoestrogens, anandamide and Vitamin A derivatives. SXR interaction with any of these compounds leads to signals inside of the cell that cause harmful agents to be broken down so that they can no longer cause cellular damage. This effect can be helpful to normal cells by removing potentially cancer-causing agents and to cancerous cells by removing the agents which can further the progression of cancer. The anti-breast cancer effects demonstrated by SXR interaction with the test compounds will be examined utilizing breast cancer cell lines and measuring effects on their growth.

This project should provide information on a new target for effective anti-breast cancer drug development, and could lead to a basic understanding of the way in which dietary factors such as phytoestrogens play a role in breast cancer prevention.

Mechanism of Radiosensitivity in Breast Cancer Cells

Xiaofei Wang, Ph.D.
The Scripps Research Institute

Radiotherapy is frequently applied to control local tumors including breast tumors. However, some cancer cells may be less sensitive to radiation and therefore are likely to survive radiation therapy. Relapse due to radioresistance of cancer cells significantly affects the success of radiotherapy and cancer patient survival. While a drug that would sensitize cancer cells to radiotherapy is greatly needed, efforts to develop such a drug have not been successful because we have just begun to understand how cells respond to radiation and become resistant. Cells respond to radiation by activating an internal protective mechanism. Findings in our laboratory indicate that a group of proteins called p38 MAP kinases belongs to this system. Using gene therapy methods to inhibit these kinase proteins makes breast cancer cells more susceptible to radiation. Thus, drugs designed to block the p38 MAP kinases may prove to be excellent sensitizers that can be used in radiotherapy. In this project, I will study in more detail how radiation turns on these proteins and how these proteins protect cells against radiation. Moreover, we will determine which molecule involved in controlling radioresistance is the best target for a radiosensitizer. A method widely used in gene therapy called recombinant adenoviral transduction will be used to introduce inhibitory genes into breast cancer cells. Cell killing by radiation will also be studied using biochemical, immunological, and cellular approaches.

This research will not only bring us vital information about the mechanism of radioresistance of cancer cells, but it will also give us a chance to work against it by designing a sensitizer. Success of this research project will thus help to enhance both the efficiency of radiation therapy and the chance for survival of breast cancer patients.

Bispecific Antibodies for Radiotherapy of Breast Cancer

Michelle D. Winthrop, Ph.D.
University of California, Davis

There is a critical need for improved treatment strategies in breast cancer for women diagnosed with metastatic disease. In such clinical settings, the current therapy options result in survival periods of less than 2 years in most cases. These breast cancer metastases are in many organs (e.g. lung and bone) and direct surgical removal or external beam radiotherapy are merely palliative. However, radioimmunotherapy, which utilizes monoclonal antibodies attached to high-energy radioisotopes, have demonstrated promise in the treatment of metastatic cancer. In addition, radioimmunotherapy is able to target multiple metastasis throughout the body in a single treatment. While excellent results have been reported using radioimmunotherapy in lymphoma, the corresponding success in solid tumors has been limited. Drawbacks of radioimmunotherapy in the treatment of breast cancer include the low tumor accumulation of the radiolabeled monoclonal antibody due to the large size of the antibody molecule as well as "bystander" radiation delivered to normal tissue during tumor localization. Thus, improvements are needed to: (1) increase the radiation delivered to tumor, while (2) decreasing the radiation to normal tissue in order to give doses of radioactive drug that will destroy all tumor cells without causing toxicity to normal tissues.

Our approach is to engineer antibody fragments to a cell surface glycoprotein, the MUC-1 antigen. This antigen is abundant on 90% of human breast cancers in forms not present in normal tissue. In preliminary work, we have generated antibodies to MUC-1, and genetically manipulated the antigen binding site (variable heavy and variable light chain) genes into single-chain antibody fragments. In this project, we will first characterize the potential utility of selected antibodies by binding studies on breast cancer cells lines and breast tumors. Those antibodies with the most favorable binding characteristics will be DNA sequenced. These will be modified and made into bifunctional agents that combine the ability to recognize the MUC-1 antigen and carry the radiometal chelate, 90Y-DOTA. Thus, the goal is to produce hybrid molecules containing both the breast cancer binding region and the radioactive chelate binding region on a single therapeutic agent. Finally, we intend to evaluate these bispecific antibodies in mice with human breast tumors to determine the appropriate phamacokinetic characteristics (i.e., blood half-life, biodistribution, tumor uptake, dosage, etc.).

We hope that our approach using genetic engineered bispecific immunotherapeutics will allows us to overcome the limitations encountered using targeted radioimmunotherapy of advanced breast cancer.

How Indole-3 Carbinol Inhibits Breast Cancer Cell Growth

Liquin Zhang, Ph.D.
University of California, Berkeley

Breast cancers differ in their responses to estrogen hormones. Approximately two thirds of the breast cancer cases are susceptible to tamoxifen, an antiestrogen. However, cancers that are estrogen receptor negative are more difficult to treat and, perhaps, prevent using tamoxifen. Moreover, patients treated with tamoxifen sometimes will have notable side effects and/or will develop resistance to tamoxifen. Thus, alternative chemopreventative and chemotherapeutic agents with novel growth-inhibiting mechanisms require research and development. Recently, our lab has discovered a novel anti-breast cancer agent, indole-3-carbinol (I3C). This compound is found naturally in vegetables such as broccoli and cabbage and can target breast cancer from a unique direction. In previous work, we found that mammary tumor cells treated with I3C show decreased proliferation rate. The production and/or activity of key cell cycle regulatory proteins, CDK6 and CDK2 are also found to be decreased upon I3C treatment. Importantly, these effects were not related to the estrogen receptor status of the cell. Thus, pending further understanding both for its mode of action and progress in drug development, it appears that I3C is a very promising broad-spectrum anti-tumor agent.

At present very little is known about how I3C works on CDK6 to block cell growth. The focus of this project is to link the cell cycle regulatory proteins with more general breast cancer cell signaling through growth factors and other stimuli. In general, these cell signaling pathways are initially triggered by binding events at the cell surface. This initiates a sequence of interacting molecules, which serves both for amplification and regulation. The end result is often to control nuclear factors that direct specific gene expression. Our specific interest is the MAPK (mitogen activated protein kinase) signaling pathway, for we have observed consistent change in MAPK protein activity in breast cancer cells by I3C treatment. In this project we plan to monitor the I3C-induced activity changes for the associated proteins in the MAPK signaling pathway in order to more clearly define the cellular targets of I3C. To accomplish this, we will manipulate the sequence of binding events in the MAPK pathway by both chemical and/or genetic procedures to see how the I3C effect becomes altered. This will be studied both in breast cancer cell lines and in tumors themselves.

Our goal is to unravel the molecular mechanism of I3C inhibition on cell cycle growth control in breast cancer. These findings will provide valuable information to facilitate the systematic therapeutic development of I3C as an efficient anti-tumor medicine.

top

Training Program Award


UCLA Biomedical Physics Graduate Training in Breast Cancer

Carolyn Kimme-Smith, Ph.D.
University of California, Los Angeles

The UCLA Biomedical Physics Graduate Program has provided graduate education in Medical Physics and Radiation Biology for 30 years. It is the third Ph.D. program in Biomedical Physics to be accredited by the American Association of Physicists in Medicine. It will award its 117th Ph.D. this spring.

At this time, its 44 graduate students are specializing in either Medical Imaging, Nuclear Medicine, Radiation Oncology, or Radiation Biology. Many of these students are interested in some aspect of detecting, treating, or preventing breast cancer, but have been unable to pursue these research interests because of a lack of funds for projects in breast cancer related research. This training grant renewal would complete the training of two Ph.D. students currently supported by the training grant and would allow two more students to be trained. In addition, the program requires courses, practical training and participation in seminar series on topics critical to understanding breast cancer, creating a group of future medical school faculty and hospital physicists who are literate in breast cancer related issues.

Those students selected for this training program will have a required course of study, preparing them for general medical physics topics, in addition to the current program's core courses, which will include a course in Breast Imaging Physics and Instrumentation, Medical Ultrasound, a Seminar in Breast Cancer Detection and Treatment, the Breast Center's Multidisciplinary Conference, and the Breast Imaging Center's Pathology-Radiology Correlation Conference. Appreciation of the clinical realities of breast cancer will be provided by requiring at least 36 hours in clinical settings, including radiology mammography reading, interventional procedures, and pathology techniques. At the end of their second year of study, but their first year on this training grant, all students will have selected an area of specialization in breast cancer related research. By the beginning of their third year of graduate study, or second year of this training program, they will be working on a specific research project in breast cancer. The two students who will continue on this grant have one and two years to complete specific Ph.D. projects. Two new students will work on early detection projects in ultrasound and digital mammography.

Many breast cancer researchers have very narrow, specialized understanding of the disease or misunderstand the relationship of their research to breast cancer detection and treatment. Our training program would provide a broad base of knowledge to allow our graduates to be resources for other researchers as well as principal investigators on their own. Our trainees will be particularly well suited to develop research and clinical approaches for improvements in breast cancer detection, for improvement of the positive predictive rate of identifying malignant versus benign lesions (minimizing unnecessary biopsy), and for improvement of the therapeutic ratio in treatment of breast cancer by radiobiological research. The graduates of this program, through their competence and broad scope of training, will have the tools to help to reduce the human and economic costs of breast cancer in California and will be the leaders of the future in breast cancer research.

top