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Organization of the Organ Systems Branch Current SPORE Programs Information for the Public PART Program Information for Applicants IntraSPORE Communications
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NORTHWESTERN UNIVERSITY MEDICAL SCHOOL
OVERALL ABSTRACT This SPORE application submitted in response to the RFA from the National Cancer Institute, will take advantage of the strengths in breast cancer research and treatment that exist at the Robert H. Lurie Comprehensive Cancer Center. The SPORE will provide the infrastructure to bring together basic scientists and clinicians to rapidly test new approaches in the prevention, early detection, diagnosis and treatment of human breast cancer. The specific themes of the SPORE are:
The SPORE application consists of six principal research projects, three career development projects, five pilot projects and four Core facilities to support the research. The principal research projects are: #1. Drug resistance to antiestrogens (PI Jordan), #2. Actions of estrogen agonists and antagonists by non-classical transcription pathways (PI Jameson), #3, Dietary and hormonal modifications of breast carcinogenesis in transgenic mice (PI Thor), #4. Estradiol levels in breast fluid, saliva and serum samples (PI Chatterton), #5. Antiestrogens and breast density in premenopausal women (PI Morrow), #6. Angiostatic therapy for breast cancer: a translational study (PI Soff). The Core facilities include: Administration, Tissue Resource, Clinical and Biostatistical Cores. The investigators selected to be part of the SPORE Program come from a variety of disciplines ranging from Clinical Programs to Chemical Engineering, Pathology and Microbiology-Immunology. To maintain meaningful communication between investigators, we will have monthly meetings of SPORE investigators, an annual retreat, monthly meetings of the Executive Committee and each investigator will be actively involved with at least one project (or core) in addition to their own. Based on the infrastructure described, our large patient population and our track record of productivity, we believe that the Robert H. Lurie Comprehensive Cancer Center is well suited for the SPORE program.
RESEARCH PROJECT 1 The development of drug resistance limits the long-term usefulness of tamoxifen for the treatment and prevention of breast cancer. One form of drug resistance that has been described is tamoxifen stimulated growth. We plan to advance understanding of drug resistance mechanisms by establishing new models of resistance to a range of clinically relevant antiestrogens. A tamoxifen resistant phenotype of the well characterized MCF-7 breast cancer cell line (ER positive, p53 wild type) has been established both in vitro and in vivo. We have also found that T47D breast cancer cells (ER positive, p53 mutant) can lose ER in vitro with estrogen deprivation but unexpectedly produce ER positive tamoxifen stimulated tumors rapidly in vivo. Since antiestrogens can cause G1 block, apoptosis and prevent angiogenesis signaling, resistant tumors must subvert these mechanisms. We propose to address the following specific aims: 1) To establish models of antiestrogen resistance in vivo and in vitro with MCF-7 and T47D cells. 2) To determine the cytocidal mechanisms of different classes of antiestrogens. 3) To determine changes in cell cycle regulation, as cells and tumors convert from antiestrogen sensitive to antiestrogen resistant. We hypothesize that different classes of antiestrogens will block the cell cycle for different times but eventually the cell cycle will return to an estrogen-like pattern once antiestrogen resistance occurs. 4) To examine the change in angiogenic signaling during the development of antiestrogen resistance. We hypothesize that cells that develop resistance in vitro may not grow in vivo because there is no selection pressure for angiogenesis. Ultimately, an antiestrogen-stimulated tumor must achieve a full complement of angiogenesis promoting genes. We propose that overall differences between MCF-7 and T47D cells may result from the loss of p53 control in T47D cells. Although it is not possible to confirm this hypothesis with a direct comparison of different cell lines, we will address the concept with MCF-7 cells, stably transfected with E6 to remove p53. A comparison of the transplantability of tamoxifen resistant T47D, MCF-7 and MCF-7 E6 cells in vitro will confirm or refute the hypothesis that drug induced tumorigenesis is enhanced through activating angiogenic pathways. The primary goal of this project is to establish a resource of well-characterized models for a range of clinically relevant antiestrogens in vivo as a first step to translating the results to clinical samples.
RESEARCH PROJECT 2 Estrogen, acting through its nuclear receptor, stimulates breast development and can enhance the growth of breast cancer. Drugs that interfere with estrogen receptor (ER) action are an important part of breast cancer treatment and are also being used for breast cancer prevention. There has been rapid progress in our understanding of how the ER acts to regulate target genes. Structural studies have provided important insights into how ligands, such as natural estrogens or the synthetic agonist diethylstilbestrol, function as agonists whereas others, such as raloxifene, tamoxifen or ICI compounds, function as antagonists in some tissues. We hypothesize that the ER modulates gene transcription by two fundamentally different mechanisms: (1) a classical estrogen response element (ERE)-mediated pathway and (2) a non-classical pathway that involves ER interactions with other transcription factors present on target genes. Agonists such as estrogen stimulate gene expression by the classical pathway, but repress gene expression through the non-classical pathway. Partial agonists and antagonists are inactive or inhibit gene expression through the classical pathway; however, they paradoxically stimulate the expression of some genes through the non-classical pathway. Unraveling the molecular basis of these ER-dependent pathways is fundamental to understanding the role of ER in breast cancer and the pharmacological actions of drugs used to treat or prevent breast cancer. In the current proposal we plan to (1) develop in vitro models of classical and non-classical ER-dependent transcription, (2) develop an in vivo knock-in mutation of ERa that discriminates its function through the classical and non-classical pathways, and (3) identify breast epithelial cell target genes in the non-classical ER pathway.
RESEARCH PROJECT 3 Hormones are known to play a crucial role in mammary cancer development. Diets rich in soy-based products or restricted in calories have been associated with a reduction in breast cancer incidence. This protective effect may be the result of modulation of estrogen metabolism or action. These factors are difficult to control and costly to study in humans. Furthermore, these studies do not lend themselves to analyses of mechanisms or gene expression using currently available technologies. The recent development of transgenic mouse models makes such clinically important studies feasible using surrogate systems. Utilizing virgin MMTV wt erbB-2 transgenic mice, which are genetially susceptible to breast cancer, we have demonstrated a co-carcinogenic and dose-dependent effect of 17-ß-estradiol administered during the risk window. Our data suggests that 17-ß-estradiol (E2) accelerates mammary epithelial hyperplasia and dysplasia in concert with downregulation of the estrogen receptor (ER), overexpression of erbB-2, activated erbB-2, EGFR and TGFa. Cancers which arise in transgenic mice given E2 supplementation (and novel tumor cell lines from these tumors) have phenotypic, biologic and molecular differences when compared to breast tumors of untreated animals. Whole mount preparations, in particular, demonstrate marked abnormalities in breast morphogenesis in parallel with these biologic changes. Our most recent (and unpublished) data shows that tamoxifen administration during the risk window (8-16 weeks) completely prevents cancer development in this model system. Soy diet, as compared to casein diet, reduces and delays breast carcinogenesis as well. Our specific aims are: 1) To confirm that dietary soy prolongs tumor latency and decreases the risk of carcinogenesis in virgin MMTV wt erbB-2 transgenic mice, which are genetically predisposed to breast cancer, using rigorously matched formulary diets (soy vs casein) with and without exogenous 17-ß-estradiol (E2) administration and tamoxifen; 2) to determine the effects of calorie restriction in virgin MMTV wt erbB-2 transgenic mice, with and without exogenous 17-ß-estradiol (E2) administration; and 3) to study the effect of 17-ß-estradiol (E2) administration (single pellet, during the risk window) on breast carcinogenesis using several additional transgenic and bi-transgenic mice including: MMTV-Wnt 1, MMTV erbB-2 X mutant p53; MMTV erbB-2 X MMTV TGFa. To our knowledge our studies are the first to link hormones to cancer in this model system and show prevention using tamoxifen. Furthermore, we are the first to suggest that transgenic models may demonstrate differences in tumor latency with a soy based (phytoestrogen rich) diet. These models will further elucidate interactions between hormones, diet and cancer and will allow molecular analyses to better define genetic and expression differences which may be associated with these factors.
RESEARCH PROJECT 4 Long-term exposure of the breast to endogenous estradiol (E2) is widely believed to be an important factor in determining a woman's risk of developing breast cancer. Previous studies have shown that breast fluid (BF) obtained from nipple aspirates has many times the concentration of estradiol in serum, and that the concentration is not reduced significantly after menopause when blood levels have declined. The purpose of the present study is to ascertain the factors and mechanisms by which estradiol levels are maintained in the breast and the biological activity associated with those levels. We will determine whether salivary estradiol, an estimate of diffusable estradiol, is more closely related to BF E2 than serum E2 in both pre- and postmenopausal women. We will determine whether adrenal androgen precursors can explain the maintenance of BF E2 in postmenopausal women. We will determine whether BF E2 or blood levels of E2 are more closely associated with the concentration of an estrogen response gene product, pS2, and to the growth factor, EGF, in BF. From the ratios of steroid products in BF we will estimate the importance of the several potential precursors of BF E2 and the role of specific cytokines for biosynthesis of BF E2. Potential feedback effects of estrogen on the levels of BF E2 will be determined in women receiving ethinyl estradiol and in women taking the estrogen antagonist, tamoxifen. For these experiments we have developed specific and sensitive assays for salivary E2, BF E2, and BF EGF and a method for separating BF products in a highly efficient manner into phenolic (estrogen), neutral (androgen), and aqueous fractions so that many more substances can be measured from a small volume of BF. Additional assays will be developed according to established protocols. We have already collected samples from premenopausal women on a dietary intervention study, and we plan to recruit premenopausal women planning to start oral contraceptive use and women who will start tamoxifen treatment as well as a group of postmenopausal women for these studies. Being able to monitor the effect on BF E2 of factors involved in its biosynthesis and in its action in human subjects will give us insight into the ways in which one of the important determinants of breast cancer risk is regulated.
RESEARCH PROJECT 5 Breast density is a major cause of the failure to detect breast cancer with screening mammography in premenopausal women. Tamoxifen is used for breast cancer risk reduction and treatment, but little is known about its effect on breast density, or the correlation between tamoxifen-induced changes in sex steroids and symptoms which decrease quality of life and limit the use of tamoxifen. The ideal agent would be one which decreased breast density without producing systemic effects. We propose to address this problem using full-field digital mammography to measure breast density reproducibly. The specific aims are: 1) to correlate changes in breast density with the hormonal changes throughout the menstrual cycle using a salivary assay for sex steroids. This study will determine whether this is a significant factor which must be controlled for in intervention studies; 2) To evaluate the alterations in breast density produced by tamoxifen, and correlate changes in steroid hormone levels with changes in quality of life; and 3) to examine the use of a topically applied antiestrogen, 4-hydroxytamoxifen, on breast density. The incidence of symptoms and changes in hormone levels will be assessed to determine the extent of systemic absorption and its clinical significance. This study will provide useful information to women about the scheduling of mammographic examinations; determine if tamoxifen reduces breast density, another potential benefit of the drug for high-risk women; and ascertain whether a local strategy of density reduction is effective without producing systemic symptoms. If so, this would offer a major opportunity to improve our ability to detect breast cancer in the female population as a whole.
RESEARCH PROJECT 6 For tumors to grow and metastasize, they induce the generation of new blood vessels, a process referred to as angiogenesis . Inhibition of angiogenesis is thus an attractive approach to inhibit cancer growth and metastasis. The angiogenesis inhibitor angiostatin is a kringle containing internal fragment of plasminogen, which has been shown to inhibit cancer growth in numerous animal models. In preliminary research, we have demonstrated the mechanism of generation of native human angiostatin, yielding an isoform referred to as Angiostatin4.5 (AS4.5) (Appendices A, B, C]. Plasminogen is first converted to plasmin by a plasminogen activator, and in the presence of a free sulfhydryl donor, plasmin undergoes autoproteolysis to yield AS4.5. Furthermore, we have shown that AS4.5 is present in plasma and other human specimens, and native AS4.5 is a potent angiogenesis and tumor inhibitor. In more recent experiments, we have now observed that AS4.5 circulates both as a monomer as well as in a complex with as yet undefined other proteins. Furthermore, in experiments in mice, and in human cancer patients administration of an "Angiostatic Cocktail," consisting of a plasminogen activator and a free sulfhydryl donor, resulted in marked increases in plasma levels of monomeric and complexed AS4.5. Administration of the Angiostatic Cocktail suppressed EOMA (hemangioendothelioma) tumors >95% in mice. In pilot experiments with patients with cancer, administration of an Angiostatic Cocktail resulted in increased plasma levels of AS4.5 from approximately 5 nM to approximately 100 nM and that this in vivo generated AS4.5 was indeed biologically active as an angiogenesis inhibitor. Most importantly, the administration of the Angiostatic Cocktail resulted in demonstrable anti-tumor activity in 4 of the first 5 patients treated. It is the purpose of this project, to further study the biochemical effects of the Angiostatic Cocktail for women with breast cancer. Studies will include development of assays for the AS4.5 monomer and complexes, as well as analysis of the biochemical and clinical responses of the Angiostatic Cocktail in the treatment of breast cancer.
ADMINISTRATIVE CORE The Administrative Core of the Robert H. Lurie Comprehensive Cancer Center Breast Cancer SPORE will provide the necessary leadership and administrative support to insure coordinated operation of the SPORE. The Core will manage all SPORE finances, facilitate interactions between the different projects and cores and will provide appropriate administrative assistance to investigators in the SPORE. The Core will coordinate SPORE monthly meetings, an annual retreat, the External Advisory Meeting and progress reports to the NCI. The Core will also be responsible for administration of the Developmental Research Program and Career Development Program including advertisement of the availability of funds, assembling an ad hoc review committee and disbursement of funds. The Administrative Core will assist the Executive and Internal Advisory Committees in the selection of mentors for the Career Development investigators. The Core consists of the PI of the SPORE, the Administrator of the SPORE, a budget manager and an administrative assistant. The Administrator is a member of the SPORE Executive Committee. These functions will ensure that the CORE maintains a central role in the SPORE Program.
TISSUE RESOURCE CORE The Tissue Resource Core of the Robert H. Lurie Comprehensive Cancer Center's Specialized Program of Research Excellence (SPORE) in breast cancer has two dedicated breast cancer treatment centers, Northwestern Memorial Hospital (NMH) and Evanston Northwestern Healthcare (ENH), dedicated to the collection, storage and distribution of human breast cancer for research. Because our institutions see large numbers of new breast cancer patients each year for surgical excision (>1100), our access to patient derived tissues for research is high. The ENH facility has nearly 5 years of experience with a prospective and retrospective ENH breast cancer bank (containing 12,000 archived cases and 450 fresh frozen samples to date), houses the national Pathology Coordinating Office (solid tumor tissue bank) for ECOG, and has participated with the UCSF and UNC SPORE programs as a sub-contracting institution. The NMH facility also has significant expertise in tumor banking through the Cancer Center's Research Histology/Tissue Procurement shared resource. This core will facilitate both the collection of fixed embedded and fresh frozen tissues as well as the acquisition and database storage of essential pathologic and clinical information needed for conducting translational research. This resource will benefit not only to the SPORE investigators, it will facilitate research activities of other scientists within and outside the parent institution. With time and accrual we will be willing to participate in NCI-supported tissue networks so that the benefits of this resource are beyond our institution. This process will be supervised by a Tissue Review and Disbursement Committee, (including physicians and pathologists), the SPORE Executive Committee and reviews by the individual Institutional Review Boards (IRBs) on an annual basis.
CLINICAL CORE The Clinical Core of the SPORE Program will be responsible for facilitating translational research through the support of clinical trials and clinical research studies. The Core will collect clinical data and patient specimen samples (e.g.serum, saliva) for SPORE investigators. Northwestern University has a National Institute of Health funded General Clinical Research Center (GCRC) located within the Northwestern Memorial Hospital for clinical trials. The Core director will be available to assist investigators interested in initiating a clinical study, both in protocol development and in IRB submission for human subjects approval. The Robert H. Lurie Comprehensive Cancer Center has an NCI approved Clinical Protocol Scientific Review and Monitoring Committee. The Core nurse will assist with actual clinical trials to insure that the clinical protocol is properly followed, provide support and education to participating patients, assist in the administration of therapy and assure timely entry of clinical data. The Core research assistant will assist with patient records and entering patient data into a database. The Core will interact closely with the SPORE Biostatistics Core in maintaining the database and with the Tissue Resource for banking of specimen samples. The Clinical Core will interface with the Cancer Center's Clinical Research Office that is responsible for the management of all cancer clinical trials.
BIOSTATISTICS CORE The Biostatistics Core will (a) develop and maintain the central database of the SPORE and (b) provide biostatistical expertise for all aspects of the SPORE. The central SPORE database will be web based and will house the clinical, tissue and laboratory data of breast cancer patients seen at the Northwestern Memorial Hospital and Evanston Northwestern Healthcare sites. Using current data collection systems as a basis, standardized procedures will be developed for collecting, checking, editing, entering, storing, managing and analyzing the SPORE data. The Biostatistics Core will interact extensively with the Clinical and Tissue Cores for the purposes of developing the database. This core will have expertise in biostatistics, database technology, systems programming and data entry. This mix of expertise will house all aspects of data management and analysis into this Core, thereby enhancing the efficiency with which SPORE informatics aspects may be viewed and dealt with on a daily basis. In addition to developing and maintaining the database, this core will be responsible for data input, data quality control, statistical analysis and statistical input to research design. This Core will be available at all times to provide input on design or statistical analysis to all SPORE investigators. Scientific input to the SPORE as a whole will be provided as direct input to the Executive Committee regarding the overall research effort of the SPORE. Scientific input by the Biostatistics Core will be provided to individual investigators of interpretation of statistical analyses and writing of manuscripts. |
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