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Vanderbilt University
Overall Abstract Principal Investigator: Carlos Arteaga, M.D. This Breast Cancer SPORE application is being submitted by the Vanderbilt-Ingram Comprehensive Cancer Center, Vanderbilt University Medical Center, and affiliated institutions. The proposal comprises clinical, translational, and basic science investigators with significant NCI support related to the focus of the application. The thematics targeted are a natural extension on the funded research of SPORE investigators and include: novel clinical trials with assessment of surrogate markers of response, the identification of markers of breast cancer risk in preneoplastic syndromes, and the discovery of proteins predictive of breast cancer invasiveness and therapeutic response using imaging mass spectrometry. Project 1 will determine the antitumor effect of OSI-774, an inhibitor of the HER (erbB) signaling network in patients with operable breast cancer and identify a molecular profile associated with evidence of response in situ. This will identify a population that can be the target of randomized trials with HER signaling inhibitors. Project 2 will determine if paclitaxel administration to patients with locally advanced breast cancer leads to tumor cell transit into mitosis, loss of proliferation, and apoptosis, and if these events predict for tumor response. Both of these Projects will use imaging mass spectrometry to discover novel protein profiles predictive of drug action in two non-overlapping patient populations. Project 3 will use imaging mass spectrometry in microdissected primary breast cancers to identify proteins that segregate DCIS from invasive cancer and tumor from non-tumor stroma. In a mouse model of breast cancer this Project will spatially profile anticancer drugs with mass spectrometric evidence of drug action on established and novel protein targets. Project 4 will determine if alterations in components of the TGFβ and EGF receptor pathways are associated with enhanced breast cancer risk in the Nashville Breast Cohort, a large cohort of women with 14 years of follow-up and with available archival material. This Project will also address for the first time in African-Americans in this cohort, whether breast cancer risk is affected by breast hyperplasia. To support these translational projects, we propose six CORES: Administrative and Outreach, Tissue, Proteomics and Emerging Technologies, Antibody Production and Characterization, Biostatistics, and Biomedical Informatics. The proposed Developmental Research (Pilot) and Career Development Programs are tightly integrated with Institutional initiatives and mechanisms that identify local research strengths as well as the best translational researchers. For these two Programs and the Tissue Core, the SPORE has harnessed significant matching support from the VICC and VUMC. The combined efforts and product of the translational and pilot projects, the career development awards, and the core resources in this SPORE application will impact the prevention, diagnosis, and treatment of breast cancer.
Project 1 The epidermal growth factor receptor (EGFR, HER1, erbB1), HER2/neu (erbB2), HER3 (erbB3), and HER4 (erbB4) comprise a family of homologous and interactive transmembrane receptor tyrosine kinases. Abundant evidence support the role of this signaling network in mammary gland development, transformation, and tumor progression. Preclinical data suggest that the simultaneous blockade of the HER2 and EGFR tyrosine kinases exhibits a synergistic antitumor effect. We have hypothesized 1) that the simultaneous blockade of HER2 and EGFR in breast cancers will result in a measurable in situ antitumor effect in patients with operable breast cancer, 2) that the inhibition of the HER signaling network may have a greater antitumor effect on the natural history of breast cancers if used in untreated early disease and/or in patients with low tumor burden. The overall goals of this translational project are: first, to determine a molecular profile in untreated, operable breast cancer that will identify candidate patients for future adjuvant therapy trials with HER (erbB) signaling inhibitors; second, to identify novel surrogate markers of drug-induced inactivation of HER signaling that will shed light on mechanisms of action or resistance; and third, by imaging mass spectrometry, explore approaches to decipher the protein markers predictive of an antitumor response to HER signaling inhibitors. To achieve these goals, we propose the following specific aims: Specific Aim 1. To determine the antitumor effect of the HER signaling inhibitor OSI-774 in patients with untreated operable breast cancer and determine a molecular profile associated with evidence of response in situ. Specific Aim 2. To determine if OSI-774 inhibits EGFR and HER2 signaling in situ and whether drug-induced changes in phospho-HER3 and of phosphorylated p21 and p27 are surrogate markers of drug action. Specific Aim 3. To determine by imaging mass spectrometry the protein profiles in pre- and post-OSI-774 tumor specimens that predict for evidence of response in situ. Specific Aim 4. To determine if a threshold level of p27 is required for the antitumor action of HER signaling inhibitors like OSI-774.
Project 2 A commonly used approach to the management of locally advanced breast cancer involves the sequential use of chemotherapy,surgery, followed by radiation and adjuvant chemotherapy. With the advent of paclitaxel, there is increasing interest in theuse of the drug in the neoadjuvant setting. Our preliminary data show that treatment of xenograft tumors in mice with paclitaxel leads to cell cycle arrest in M-phase that is accompanied by phosphorylation of Bcl-2 and other mitotic epitopes. Further, in the majority of epithelial tumor cells lines examined, the paclitaxel-induced mitotic arrest is transient and cells can exit from mitosis with a 4N DNA content. If the paclitaxel-exposed tumor cells are deficient in G1/S checkpoint response, in particular the cyclin-dependent kinase inhibitory activity of p21cip1/waf1 (p21), we have shown that they can inappropriately enter S-phase with a 4N DNA content. Our laboratory and others have demonstrated that these biochemical events are associated with decreased proliferation and increased apoptosis in the presence of paclitaxel. Thus, these biochemical events may serve as molecular markers of paclitaxel chemoresponsiveness in human tumors. We hypothesize that those patients with locally advanced breast cancer who show the greatest degree of M-phase arrest, Bcl-2 and Cdc25C phosphorylation, and low or absent p21 protein after paclitaxel treatment will be the ones who have the highest rate of complete pathologic response. Moreover, we postulate that we can find additional predictive markers of paclitaxel response by examining protein profiles of pre- and post-paclitaxel exposed tissue using the power of mass spectrometry. In sum, we hypothesize that patients with tumors that show the greatest degree of change in select molecular markers and overall protein expression patterns from pre- to post-paclitaxel treatment, will be those that go on to a complete pathologic response. These interrelated hypotheses will be tested through the following specific aims: Specific Aim 1: To predict the degree of tumor response from paclitaxel-mediated changes in markers of cell cycle position, proliferation, and apoptosis that result from paclitaxel administration in patients with locally advanced breast cancer. Specific Aim 2: To predict the degree of tumor response from paclitaxel-mediated changes in p21 protein level and/or phosphorylation in patients with locally advanced breast cancer. We will (a) determine if p21 levels or phosphorylation increase after paclitaxel administration in patients with locally advanced breast cancer and (b) evaluate upstream signaling pathways required for p21 increase and phosphorylation after paclitaxel administration in breast epithelial cells. Specific Aim 3: To evaluate protein expression profiles by mass spectrometry (MS) in biopsy material collected pre- and post-paclitaxel treatment. We predict that patients with pathologic complete response will demonstrate a greater change in protein expression patterns than patients without pathologic complete response. We postulate that there is a molecular profile that can be identified from biopsies collected pre- and post-paclitaxel treatment that will predict which patients will show the greatest degree of response to paclitaxel as measured by the degree of pathologic response at time of definitive surgery. Further, we will gain significant insight to alternative mechanisms of paclitaxel anti-tumor activity and resistance through our mass spectrometry-based analyses of protein profiles from pre- and post-paclitaxel biopsy material.
Project 3 Mass spectrometry (MS) is a powerful tool for the rapid and precise identification of proteins, either in an isolated, purified state or in situ in tissues and cells. Recent advances in protein molecular profile analysis and imaging mass spectrometry allow direct analysis and spatial localization of tumor specific biomarkers in thin tissue sections. Furthermore, MS techniques have been devised to study complex molecular interactions directly in cells, and thus elucidate patterns of signal transduction and other regulatory functions of proteins. Direct analysis of the whole proteome by mass spectrometry has the potential to rapidly address the global changes that control the heterogeneous biology of breast cancer. Therefore, we have chosen to address four important questions in this project: 1) the transition of in situ to invasive breast cancer, 2) the spatial correlation in tissues of anticancer drugs and surrogate markers predictive of response to therapy, 3) the identification of novel protein expression patterns that predict response to therapy, and 4) the identification of proteins in signaling complexes that are altered during breast cancer progression and which are targeted by molecular therapies. To address these questions we have formulated the following specific aims: Specific Aim 1. To determine the protein expression profiles of in situ and invasive breast cancers and to apply new methods of analysis for proteomics expression data that identify expression differences predictive of biologic behavior. Specific Aim 2. To determine if spatial profiling of anticancer agents in mammary tumors corresponds to target protein changes predictive of an antitumor response. Specific Aim 3. To discover by mass spectrometry novel proteins or protein profiles predictive of therapeutic response in mammary tumors. Specific Aim 4. To identify changes in signaling protein complexes induced by anti-signaling therapies using Direct Analysis of Large Protein Complexes (DALPC) and tandem MS.
Project 4 We will study molecular and histologic markers of breast cancer risk in women with benign proliferative breast disease. Specific markers to be evaluated include the T29®C polymorphism of the TGF-β1 gene, the type I and II receptors for the TGF-β cytokines, the phosphorylated Smad2 intracellular signaling molecule, and the ErbB-1 and ErbB-3 receptors of the epidermal growth factor receptor family. We will also study the effect of epithelial hyperplasia lacking atypia on breast cancer risk among African-Americans. We are currently conducting a large retrospective cohort study of women who underwent benign breast biopsy. Paraffin embedded tissue from the entry biopsy of these patients is available. By the end of this project we project that we will have observed 781 breast cancer cases during follow-up among 16,846 study subjects in this cohort. We will expand this cohort to include women from Metro General/Hubbard Hospital in order to increase the number of African-American women available for study. We will conduct a series of nested case-control studies on these women. Women who develop breast cancer on follow-up will be the case patients in these studies. Two controls will be selected for each case matched on age and year of biopsy. PCR and immunohistochemical methods will be used to study gene polymorphisms and abnormal protein expression, respectively. Conditional logistic regression analysis will be used to assess the individual and combined effects of molecular, histologic and epidemiologic variables on breast cancer risk. This project will permit the combination of modern methods in molecular biology, pathology and epidemiology to assess potentially powerful new markers of breast cancer prognosis, and may lead to important advances in the prevention and treatment of this disease.
Core 1 The Administrative and Outreach Core is responsible for managing the SPORE resources, and facilitating communications between the SPORE components, other collaborators, and with other SPOREs and the NCI. This is accomplished through a series of oversight committees, organized administrative and scientific meetings of SPORE investigators, institutional representatives and external reviewers. In addition, the outreach goals of the Breast Cancer SPORE will be coordinated through this core.
Core 2: Tissue Core The specific aims of the Tissue Core are 1) To collect, bank, and distribute for research purposes human breast tissues to investigators in the VICC BREAST CANCER SPORE; 2) To perform quality control to ensure that the relevant tissue is supplied to the researcher, and that tissues are suitable for the planned research (not necrotic or involved by unsuspected disease processes); 3) To protect patient confidentiality through use of a pre-surgical consent form that specifically addresses use of extraneous tissue for research purposes and through de-identification of specimens, or through anonymization of specimens if informed consent is waived; 4) To conduct with the Biomedical Informatics Core, an informatics strategy for networking of requests, specimen tracking, and for extraction of de-identified data relating to specimens of interest. We will help identify suitable specimens from the pathology archives, either by diagnosis, histologic features, demographics, clinical features, or other outcomes data, and will develop and maintain a centralized database of prospectively collected and banked specimens; 5) To provide expertise in developing, performing, and evaluating immunohistochemical (IHC) and FISH assays for BREAST SPORE investigators, including IHC on tissue microarrays; 6) To provide laser microdissection services to the BREAST SPORE investigators. The establishment of a BREAST CANCER SPORE Tissue Core will build upon two already established mechanisms for tissue collection: the Tissue Acquisition Shared Resource of the Vanderbilt-Ingram Cancer Center and the newly-awarded site for the Cooperative Human Tissue Network (CHTN). Mechanisms for collection of human breast tissue and quality assurance are already in place and are used on a daily basis.
Core 3 The role of the Proteomics and Emerging Technologies Core (PETC) is to provide cost effective, state-of-the-art instrumentation and techniques to the research team for the identification of proteins and protein complexes involved in the initiation, maintenance and propagation of breast cancer. The Core is directed by Dr. David B. Friedman and co-managed by Dr. David L. Hachey, with administrative oversight by Dr. Richard M. Caprioli. The PETC will offer users an array of techniques for separation of proteins obtained from tissues and cell culture by 1-D and 2-D polyacrylamide gel electrophoresis (PAGE) using high-sensitivity detection with fluorescent tags. The Core has a dedicated ion trap LC/MS instrument equipped with an automated capillary HPLC inlet. A matrix-assisted laser desorption ionization (MALDI) mass spectrometer is available for use by the PC in Dr. Caprioli's research laboratory. Additional mass spectrometry facilities are available for use on an ad hoc basis in the shared instrument center directed by Dr. Hachey. Laboratory personnel assist users in developing protein isolation and separation techniques, assist in experimental design, develop standard operating procedures, maintain QC records on instrument performance and maintenance history, perform routine assays for investigators and train students and fellows in the theoretical and practical aspects of proteomics. The Core is run as a limited-access facility in which users are expected to prepare their own samples and subsequently bring them to the PC for separation and analysis. The Core has dedicated operators for protein separations and mass spectrometric analysis who are available to assist users as necessary. Administrative staff monitors the use of the instrument facilities by investigators and prepares reports on utilization for the SPORE director. The PI's of Project 1, 2 and 3 have requested proteomics services. The most frequently requested services are those related to identification and characterization of proteomes by 2-D PAGE and MS, analysis of protein expression by MALDI profiling, and protein expression using multispectral analysis of differentially labeled proteins.
Core 4 The mission of the Antibody Production and Characterization Core (APCC) is to produce and characterize antibodies for research use by members of the Breast Cancer Spore program. Core personnel are skilled in immunoassay development, hybridoma antibody and phage-display recombinant antibody technology, and methods for high-throughput antibody detection and characterization. The core uses traditional hybridoma technology to develop mouse and rat monoclonal antibodies and employs large rodent (~2.9 x 109 members) and human (~5.9 x 109 members) phage-displayed antibody libraries to obtain antigen-specific antibodies. The APCC has robotic systems (e.g. Genetix Q-Pix Colony-picker and Robbins HTS), 384-well visible and fluorescent plate readers, an imaging system (BioRad FluorS Max2), and a BIAcore 2000 for rapid antibody detection and characterization. The APCC has generated monoclonal and recombinant antibodies to more than seventy different antigens. The APCC has used phage-displayed recombinant antibody libraries to generate phospho-specific single chain fragment variable (ScFv) antibodies and will use this approach to address the specific aims of this grant. The Specific Aims of this proposal are initially to:
Core 5 The purpose of the Biostatistlcs Core is to provide professional expertise in statistics for all Vanderbilt University Breast Cancer SPORE projects, investigators and participants. Functions provided by this core include development of experimental designs, power analysis and sample size estimation, data acquisition and database development, statistical analysis and interpretation of findings, and collaboration on presentation of results. To achieve these functions, the core director and core biostatisticians are constantly available to investigators, and are in regular contact with the project and core leaders. The primary objectives of the Biostatistics Core are:
The Biostatistics Core SUPPOlt is required in all Breast Cancer SPORE studies. Core personnel have worked and will continue to work closely with project leaders to assure that the Core provides state-ofthe-art statistical support.
Core 6 The mission of the Biomedical Informatics Core is to provide professional expertise in informatics to enable interpretation of results generated by matrix assisted laser desorption-ionization mass spectrometry (MALDI-MS) imaging high throughput technologies to study the biology of breast cancer and the molecular basis of its response to therapy. Protein profiles are important as they are representative of the functional entities that define the phenotype of the cell. Primary objectives for the core include:
The ultimate goal is to integrate expert knowledge, massive data sets, and statistical analysis into a single framework with data mining tools that we can use to derive molecular mechanisms for diagnosis, prognosis, and prediction.
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