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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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BAYLOR COLLEGE OF MEDICINE
Project 1 De novo and acquired resistance to endocrine therapy are major clinical problems in the treatment of breast cancer. Two new findings from our laboratory strongly suggest that cross-talk between the estrogen receptor (ER) and the growth factor and stress signaling pathways is an important component of resistance. First, we have found in our clinical tumor bank samples that overexpression of the ER coactivator AIB1 (SRC-3) is required for the tamoxifen resistance associated with HER-2 amplification - and AIB1 is activated by growth factor signaling via ERK 1,2 MAP kinase. Second, we have discovered in our preclinical xenograft model system that acquired resistance to tamoxifen or to estrogen deprivation is associated with activation of two stress-signaling molecules, jun N-terminal kinase (JNK) and p38 MAP kinase, which can directly or indirectly modulate ER activity. Indeed, we find that drugs targeting these pathways, already being developed for other clinical uses, not only block signaling through these kinase cascades, but also abrogate the ER phosphorylation induced by them. Our overall hypothesis is thus that both growth factor and stress MAPK pathways contribute to the failure of endocrine therapy by phosphorylating and activating ER and/or key ER coregulatory proteins like AIB1. These pathways may thus provide predictive markers of clinical endocrine resistance. Furthermore, we suggest that the onset of resistance might be delayed or reversed by inhibitors of these pathways, resulting in more effective combined endocrine therapy. We therefore propose: 1) To determine the role of EGFR/HER-2 signaling and the ER coactivator AIB1 in de novo and acquired resistance to endocrine therapy in tumors over expressing HER-2; and 2) To define the role of stress signaling pathways in acquired endocrine resistance in tumors expressing normal levels of EGFR/HER-2. We will determine whether these mechanisms of resistance are specific for certain types of endocrine therapy with different mechanisms of action (tamoxifen, estrogen depletion, pure antiestrogens), and whether combining these ER-targeted therapies with inhibitors of these MAPK pathways or of AIB1 itself offers a promising new treatment strategy, allowing rational design of future clinical trials of this approach. Finally, we will confirm the importance of these pathways in clinical samples, and will also investigate whether these signaling molecules might serve as clinically useful predictive biomarkers for endocrine response or resistance in patients. Project 2 Ductal carcinoma in situ (DCIS) is in itself an essentially harmless disease, but it gives rise to about 90% of invasive breast cancers (IBCs), which are potentially lethal. DCIS is currently defined by its histological features, and its prognosis is imprecisely estimated from epidemiological evidence. While lesions within general categories look alike under the microscope, only a subset progresses to IBC, emphasizing that there are underlying biological abnormalities causing some to remain stable and others to progress. The long-term goal of this proposal is to identify these abnormalities and begin to assess their clinical usefulness. Unique resources, exciting preliminary data, and an experienced team of investigators have been assembled to achieve this goal. The resources and data include an unrivaled bank of fresh-frozen DCIS (n = 25), as well as matched cases of normal breast and IBC for comparison, which have been evaluated for the expression of over 12,000 annotated genes using cDNA expression arrays (Clontech and Affymetrix). Analysis of this data has identified clusters of genes that may be important in the evolution of DCIS. The importance of selected genes will be confirmed and validated using another unique resource -- hundreds of samples of human DCIS with clinical follow-up, as well as other relevant types of breast tissues, assembled in tissue arrays. Aim 1 will identify genes important in the early development of DCIS by comparing expression profiles between normal breast and DCIS. The relevance of differentially expressed genes will be validated by immunohistochemistry (IHC) or RNA in situ hybridization (ISH) on tissue arrays of normal human breast and DCIS. Aim 2 is designed to identify families of biologically related DCIS through hierarchical clustering of expression array data, and to determine if they are prognostically distinct. This will again be validated by IHC/ISH on tissue arrays of DCIS from breasts with vs. without synchronous IBC. Aim 3 will compare gene expression patterns between DCIS and IBC to identify potential invasion-related genes , which will be evaluated for their prognostic significance using a case-control strategy similar to Aim 2. Finally, Aim 4 will determine whether a few of the most highly prognostic genes identified in previous aims are directly involved in causing tumor invasion/progression . We will transfect the genes into human MCF10AT cells to determine how they change the expected proportions of DCIS and IBC arising in mouse xenografts. Understanding DCIS at a fundamental genetic level may enable more accurate assessment of risk of progression, individualization of therapy for DCIS and, most important, identification of specific defects that can be targeted therapeutically to prevent the development and progression of DCIS. Project 3 Recent clinical trials have shown that antiestrogens can reduce the risk of invasive breast cancer in high-risk women without existing cancer. However, antiestrogens do not reduce the incidence of estrogen (ER) receptor negative breast cancers in these women. Thus, there is an urgent need to identify and test agents that will prevent the development of ER-negative breast cancer. While ER-negative breast cells do not respond to estrogen, many do require growth factors such as EGF, TGFß, and IGF-I, so that inhibitors of signal transduction from these growth factors could be prevention candidates. A particularly promising agent is Iressa, which inhibits the tyrosine kinase activity of the EGF receptor. This drug suppresses the growth of many cancer cells in vitro , including breast cancer cells. Phase I and II clinical trials of Iressa to treat cancer have shown dramatic responses, and have also shown that Iressa is well tolerated with minimal toxicity. Our preliminary studies demonstrate that Iressa blocks signal transduction and inhibits growth in normal as well as malignant breast tissue, and suppresses the formation of mammary tumors in transgenic mice. We now propose three Aims testing the hypothesis that Iressa will prevent the development of ER-negative as well as ER-positive breast cancer in animals, and will inhibit signal transduction and suppress breast cell proliferation in humans. 1) Does Iressa prevent ER-negative breast cancer induced by activation of peptide growth factor receptors? We will treat both MMTV-TGF a and MMTV-erbB2 transgenic mice with Iressa or vehicle, compare the time to tumor formation, tumor multiplicity, and tumor growth rate, and investigate how Iressa affects proliferation, apoptosis, and activation of downstream signaling intermediates in mammary tissue. 2) Does Iressa prevent both ER-positive and ER-negative breast cancers arising in P53-null mammary glands? We will use mice receiving mammary gland transplants from P53 null mice?the mammary tumors that develop are approximately 20% ER-positive and 80% ER-negative?to determine whether Iressa suppresses both tumor types, and whether the combination of Iressa plus tamoxifen suppresses tumor development more effectively than either treatment alone. 3) Does Iressa inhibit signal transduction and suppress breast cell proliferation in women at high risk of breast cancer? We will conduct a Phase I/II chemoprevention trial in high-risk women, determining whether Iressa decreases markers of proliferation and activated signal transduction in breast epithelial cells obtained by core needle biopsy, and comparing two doses of Iressa for their effectiveness as well as their safety profile and toxicity in these women. These studies will provide the preclinical and clinical foundation to develop tyrosine kinase inhibitors as cancer preventive agents in future Phase III breast cancer prevention trials. Project 4 Recently we discovered a single point mutation in estrogen receptor a , replacing lysine 303 with arginine (K303R ER a ), in about a third of clinical premalignant breast ductal hyperplasias. Transgenic mice expressing the mutation in the mammary gland likewise develop highly proliferative ductal hyperplasias. The mutant ER is hyper-sensitive to estrogen, suggesting that it might drive inappropriate growth and therefore propel these hyperplasias toward malignancy, invasion, and ultimately metastasis. Indeed, we found that the K303R ER mutation is present in 38% of node-negative breast cancers, but in 85% of node-positive breast cancers. The K303R ER a mutation is thus the most frequent mutation reported in invasive breast cancer, indicating that it could be a marker of aggressive metastatic behavior. We hypothesize that activation of ER a by mutation at the K303 site is a "permissive" event occurring early during breast cancer evolution, and that its hypersensitive phenotype provides a proliferative advantage which could favor tumor progression and metastasis. Because the mutation is seldom if ever found in normal tissue, it could provide a highly specific treatment target. We therefore propose: 1) To determine whether the K303R ER a mutation influences the metastatic behavior of breast cancer cells . Snp analysis will be used to sequence the mutation in paired primary and lymph node or distant metastases from breast cancer patients. The role of the ER mutant in metastatic behavior will be examined both in vitro and in xenograft models. 2) To determine whether the mutation alters outcome of hormonal therapies , in specimens from a clinical trial and in a bitransgenic mouse model. 3) To determine how the mutation might be exploited as a target for new therapeutic strategies . Using peptide display technology, we will develop peptide antagonists to ER binding of the coactivator SRC-2, which we have found to be greatly enhanced by the ER mutation, and will assess the ability of these peptide antagonists to inhibit ER function and block the growth of breast cell lines overexpressing the mutant ER.
Project 5 In lieu of prevention and early diagnosis, optimal treatment strategies are the most important variable in effective control of breast cancer. We hypothesize that patterns of gene expression exist that will distinguish primary breast cancers that are sensitive or resistant to specific chemotherapies. This project will establish gene expression parameters of response for two mainline chemotherapies for breast cancer: Taxotere (T), and Adriamycin (doxorubicin) plus cyclophosphamide (AC). We will use cDNA microarrays and allied technologies, e.g. QRT-PCR or immunohistochemistry (IHC), to identify and confirm patterns of gene expression associated with sensitivity or resistance to these two therapies. Our preliminary data strongly suggests that differential gene expression correlates with T response. Sensitive tumors have higher RNA expression of mitochrondrial proteins, inflammatory-response proteins, and motility-related microfilament proteins. Resistant tumors show elevated levels of ß tubulin, increased proliferation (KI67), and elevated oncogene expression levels. We will extend these preliminary expression array results by analyzing additional specimens, both sensitive and resistant tumors, and confirm key expression differences by IHC and QRT-PCR. As this profile differs from the expected profile of general chemoresistance, we hypothesize that this molecular portrait is specific for T sensitivity and resistance. From frozen core biopsies taken from the primary tumors before treatment, we will next prospectively validate in a randomized clinical trial the expression profile for T sensitivity and identify a parallel expression profile for AC chemotherapy sensitivity . From these expression array results, genes whose expression could distinguish between T and AC sensitivity may be identified. We then plan to develop a panel of specific predictive markers for T and AC, and validate their effectiveness in predicting responses on paraffin-embedded archival tissue from patients treated with either regimen . We will validate 5-10 of the most relevant and specific markers for each regimen. This project will identify, confirm, and validate prospectively and retrospectively, key genetic pathways involved in the sensitivity and resistance of the two main treatment regimens in breast cancer, T and AC. Identification of these pathways may lead to optimization of treatment for individual patients, and thereby reduce over-treatment, unnecessary toxicity, and overall cost. Core A The National Breast Cancer Tissue Resource has been a core component of this SPORE program since its inception in 1992. This Resource has a long track record of prioritizing requests and distributing tissue to qualified breast cancer researchers both within the SPORE network and outside -- it has distributed more than 10,000 human breast tissues to 78 investigators throughout the world. Despite disastrous flooding last June that destroyed more than 65,000 specimens, this Tissue Resource currently maintains more than 11,000 permanent sections that were created from frozen tissues, and continues to provide tissues to researchers. The Tissue Resource will now be rebuilt and expanded using retrospectively identified paraffin blocks from patients who were previously diagnosed with breast diseases, along with prospectively collected paraffin blocks and frozen tissue from patients with newly diagnosed breast cancer. It is anticipated that approximately 20,000 retrospectively identified paraffin blocks will be added to the inventory during this funding period. Paraffin blocks from an additional 2,000 newly diagnosed patients and frozen tissues from at least 625 of these patients will be prospectively obtained with appropriate informed consent. The Tissue Resource will also receive, process, store, and deliver tissues that are collected as part of clinical trials conducted as SPORE projects, for which patients will be enrolled at Baylor College of Medicine and at collaborating institutions in several other cities, as indicated in each project. All components are in place to maintain this tissue resource, store the tissue, and link the tissue with appropriate pathological, clinical, and family history data. There is excellent informatics and database support for this Resource in the Biostatistics and Data Management Core, with additional expertise in the remainder of the Breast Center. Core B The Biostatistics and Data Management Core is responsible for providing (1) coordinated, centralized database management support to SPORE Projects and Cores, and (2) comprehensive biostatistical consultation, data analysis, and reporting. A well functioning core will be absolutely critical to the success of the SPORE program since all five projects in this proposal have substantial needs for biostatistical support and this support is not available elsewhere. Centralizing biostatistical support within the Core will ensure that the biostatisticians will be completely familiar with all aspects of the projects. The SPORE program will also benefit greatly from having a dedicated and experienced team of analysts devoted to analysis of microarray experiments. For all types of research data (human, mouse, microarray, etc), the Core will execute planned analyses in an orderly and timely way. Core personnel can also help the investigators design new studies and test new hypotheses that may arise by cross-fertilization of these related projects. Finally, centralized database management will be critically important to the two projects proposing multi-center clinical trials (Projects 3 and 5) and to the Tumor Bank (Core A). Core C The purpose of the Pathology Core is to provide comprehensive histopathological support to all the five projects of this proposal. The services will include routine tissue preparation, staining, laser capture microdissection, and immunohistochemistry. In addition, two very important areas of specialization of the Core will be preparation of tissue arrays, and development of immunohistochemistry assays for new antibodies. All these tasks play a central role in the experimental designs of all the projects, and the latter two are critical to the feasibility of some of the aims in individual projects. Overall this core will process 10,000 human and animal tissue samples and process and interpret 20,000 slides. We believe that this core is essential to ensure cost efficiency as well as providing the best quality of results for this large workload. We have the necessary laboratory space, equipment, and human resources in terms of expert pathologists and histotechnologists to successfully manage this work. Core D Our Breast SPORE consists of five full research projects as well as developmental projects, career development, and specialized core resources. An Administrative Core is needed to efficiently utilize administrative personnel and to provide common services for these projects and cores. Dr. C. Kent Osborne, as Principal Investigator of the SPORE, will direct this Administrative Core. The Core provides a pool of services which are common to all components of the SPORE, including: administrative processing of review and funding of Developmental Projects, recruitment of Career Development candidates, financial administration of grant funds, organization of conferences and seminars, manuscript preparation, coordination of travel, report preparation, general clerical support, processing of IRB protocols, and assurance of compliance with all NIH and institutional grant regulations. The Core will also coordinate the services of our Internal Advisory Committee, and arrange for the visits of our External Advisory Committee members to review our progress and make recommendations. In summary, consolidation of common support and administrative functions relieves individual projects of many minor but important tasks, and assures quality control in record-keeping, services, and compliance issues. Core personnel are highly experienced, and the Core provides well-organized and cost-effective support to all components of the SPORE. Developmental Projects (Chamness) In order to enable SPORE investigators to rapidly develop new research opportunities which could translate into early benefits for breast cancer patients, and to allow for exploration of new techniques which may require substantial efforts but which are nevertheless not ready for full scale multi-year research funding, we have developed this SPORE Developmental Research Program. Career Development (Chamness) The purpose of this program is to award and support promising investigators who will participate in translational breast cancer research projects. Two awards are proposed to support either MD or PhD candidates. Candidates will be selected based on their previous accomplishments and their desire to pursue a career in academic breast cancer research. While our primary focus will be to support promising young investigators, it is possible that more established investigators may also be appropriate for support. LIST OF CURRENT INVESTIGATORS C. Kent Osborne, MD (Professor and Director of the Breast Center at Baylor)
D. Craig Allred, M.D. (Professor)
Powel H. Brown, MD, PhD (Associate Professor)
Gary C. Chamness, Ph.D (Professor)
Jenny Chang, M.D. (Associate Professor)
Richard M. Elledge, M.D. (Associate Professor)
Suzanne A.W. Fuqua, Ph.D. (Professor)
Susan G. Hilsenbeck, Ph.D. (Professor) Michael Lewis, PhD (Assistant Professor) Syed Mohsin, M.D. (Assistant Professor) Peter O'Connell, Ph.D. (Professor)
Rachel Schiff, PhD (Assistant Professor) |
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