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Project # Year
of Award
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Abstract Text (Official)
R21 CA107932-01 2004 DE VERE WHITE, RALPH W. UNIVERSITY OF CALIFORNIA DAVIS Yeast Assay for p53 Molecular Analysis in Bladder Cancer
Bladder cancer is the fifth most frequently diagnosed tumor in the United States, with more than 57,400 new cases and 12,500 deaths annually. Eighty-five percent of these deaths come from the 25% of patients who present with muscle-invasive disease. Reduction in morbidity and mortality will come from improving outcome in this group of patients. Having rapid access to molecular analysis data and knowing its relationship to clinical outcome could be invaluable to patient management and treatment. The long-term objectives of this R21/R33 application are to 1) standardize the protocol for an archival-based yeast functional assay (aYFA) that will lead to broad clinical utilization, and 2) determine what clinical relevance molecular analysis may provide when detecting and evaluating p53 functionality in bladder cancer. A yeast functional assay is an existing methodology that detects p53 mutations in human cancers. In this assay, both p53 expression plasmids and reporter plasmids are co-transfected into yeast. The expressed wild-type (wt) p53 is able to bind to a p53-response element, whereas mutant p53 lacks the ability to bind. This binding and lack of binding leads to color differences in transactivational activity. Use of this current version of the yeast assay has one serious drawback: it was designed for the study of mRNA from cell lines and fresh-frozen tissue. Unfortunately, in large clinical trials, fresh-frozen tissue is rarely available for comparing genetic markers to patient outcome. Therefore, an archival-based yeast functional assay could prove to be extremely suitable for rapid, accurate, and treatment-relevant molecular analysis in a wider bladder cancer population. The specific aim of the R21 phase of this grant is to optimize the technology and methodology for a broader utilization of the archival-based yeast functional assay in the clinical setting. The specific aim of the R33 phase of this grant is to determine the clinical relevance of different p53 mutations (alterations and functionality) in bladder cancer. It is expected that the archival-based yeast functional assay technology will impact the molecular analysis of bladder cancer by proving to be both rapid and clinically relevant. The archival-based yeast functional assay technology is innovative in that it would give clinicians, regardless of location, the ability to obtain molecular analysis information that could prove valuable to patient outcome prior to actual treatment initiation. Currently, with fresh-frozen tissue required to perform the standard yeast assay, timely accessibility of individual patient molecular data for the clinician is virtually impossible to obtain. This makes the standard yeast assay less than optimal for widespread use and subsequently hampers future research endeavors. This grant proposes to compare p53 molecular analysis data and its relationship to patient clinical outcomes in bladder cancer patients. Three groups of patients that have already been reported on will form the basis of the R33 pilot study. The archival-based yeast functional assay technology will be used in testing bladder cancer specimens (cystectomy and TURBT) and then comparing their p53 status to patient clinical outcomes. The results obtained during the period of this grant will form the basis for future studies that should affect treatment strategies for this disease.
R21 CA101171-01A1 2004 DOVICHI, NORMAN J UNIVERSITY OF WASHINGTON Single Cell Protein Fingerprinting - Barrett's Esophagus
This proposal combines the efforts of researchers in the Departments of Chemistry and Pathology at the UNIVERSITY of Washington and at the Fred Hutchinson Cancer Research Center. These researchers will perform a pilot study to evaluate new technology for cancer research. This technology produces protein fingerprints from single cells and will be evaluated for the study of cells isolated from biopsies obtained from patients in the Seattle Barrett's Esophagus Cohort. This Application of Innovative Technologies for the Molecular Analysis of Cancer proposal is the first study of cell-to variation in protein expression in human precancerous tissues. In the R21 phase of this project, we will demonstrate the generation of a two-dimensional capillary electrophoresis fingerprint of the proteins from single cells isolated from an esophagus tissue biopsy. Once we demonstrate that we can successfully analyze a single cell isolated from the solid tissue, we will proceed to the R33 phase of the project. During that phase, we will perform several experiments of interest to cancer biologists. We will dissect and generate two-dimensional protein fingerprints from 20 cells from a single crypt isolated from a biopsy. This study will provide understanding of the change in protein expression during development of a stem cell into its differentiated form. We will generate fingerprints from three crypts obtained from a single biopsy. These crypts will be closely related and the study will provide information of the intraclonal variation in protein expression. We will generate fingerprints from cells isolated from crypts obtained from three biopsies from the same patient. This study will provide information on the interclonal variation in protein expression. We will generate fingerprints from 10 patients, 5 negative for dysplasia and 5 with high grade dysplasia. This study will provide information on the differences in protein expression between patients at low risk and patients at high risk for development of esophageal adenocarcinoma.
R21 CA107858-01 2004 MAITRA, ANIRBAN JOHNS HOPKINS UNIVERSITY A Sequencing Microarray for Mitochondrial Mutations
Pancreatic cancer is a lethal disease, with the vast majority of patients presenting in an advanced, inoperable stage. There is an urgent need to develop sensitive and specific molecular biomarkers for early detection of pancreatic cancers. Somatic mitochondrial DNA mutations are common in cancers, but their usefulness as a biomarker for early diagnosis has been impeded by low-throughput and lack of adequate sensitivity of direct sequencing technologies. An oligonucleotide-based mitochondrial sequencing microarray ("MitoChip") has been designed that can sequence approximately 29kb of double stranded DNA in a single assay. Both strands of the entire human mitochondrial coding sequence (15,451bp) are arrayed on the MitoChip; both strands of an additional 12,935 bp (84% of coding DNA) are arrayed in duplicate, providing internal validation of sequence data. 1.6 million bp of mitochondrial DNA have been sequenced, with intra- and inter-chip sequence reproducibility of >99.99%. In serial dilution experiments using mixed normal and tumor DNA, the MitoChip was able to detect an aberrant clonal population in as much as 50-fold diluted samples. The eventual goal of this proposal is to enable early diagnosis of pancreas cancer using endoscopically-obtained pancreatic juice samples. The central hypothesis is that array-based sequencing can reliably detect mitochondrial DNA mutations from pancreatic juice samples containing shed neoplastic cells. The R21 phase will therefore (a) identify the frequency and patterns of any confounding tissue-specific coding sequence variations that may occur in non-neoplastic pancreata compared to lymphocyte DNA, (b) determine the frequency of mitochondrial coding sequence mutations in a series of primary pancreatic cancers (using matched lymphocyte DNA as control), and (c) establish the conditions for long PCR amplification and MitoChip assay from pancreatic juice DNA. In the R33 phase, endoscopically obtained pancreatic juice from patients with pancreatic cancer, non-neoplastic pancreatic disease (e.g., chronic pancreatitis), and non-diseased controls (n=40 in each category) will be analyzed for mitochondrial DNA mutations. Juice samples from a specific subset of patients with localized, node-negative pancreatic cancer will also be examined to confirm the utility of the MitoChip in detecting early stage, and hence potentially curable, disease. This study will serve as a model for array-based diagnosis of cancer in clinical samples.
R21 CA107966-01 2004 SCHROEDER, JANE CLOWNEY UNIVERSITY OF NORTH CAROLINA CHAPEL HILL Epidemiology of Atypical Epstein-Barr Virus in Lymphoma
Traditional assays to identify Epstein-Barr virus (EBV) associated lymphomas detect viral gene products that may not be expressed when the EBV genome is rearranged or partially deleted. Recent evidence of atypical EBV in lymphomas that were EBV-negative by "gold standard" EBER in situ hybridization assays suggests EBV may contribute to a larger proportion of lymphomas than previously assumed. The goal of the proposed study is to develop and apply a novel set of cost-effective and reliable assays to clarify the presence of EBV in non-Hodgkin's lymphoma (NHL). Specific aims for the R21 phase are to 1) optimize quantitative real-time PCR assays targeting five disparate but highly conserved EBV genes plus the defective W-Zhet rearrangement, 2) use these assays to estimate EBV viral load in archival tissues comparable to samples available for population-based research, 3) use in situ assays to localize infection to neoplastic cells, 4) identify a subset of assays and viral load cut points that reliably identify cases EBV, or remnants of EBV in neoplastic cells, and 5) compare PCR results with EBER-ISH to determine whether traditional methods misclassify EBV-associated cases. Specific aims for the R33 phase are to 1) use the optimized battery of assays to identify neoplastic EBV in archival sections from 163 unselected NHL cases, 2) compare results with EBER-ISH to determine whether the new assays enhance detection of EBV-related tumors, 3) describe relations between somatic mutations and typical and atypical EBV-positive cases and 4) conduct a preliminary analysis of risk factors for typical and atypical EBV-positive NHL. Quantitative PCR assays may be cost-effective for large-scale research, but lack of atypical EBV-positive lymphomas would substantially discount hypotheses regarding hit-and-run pathogenic mechanisms resulting in EBV deletion or rearrangement. Evidence that the novel assays detect new EBV-associated cases would support a large population-based study to confirm results. Improved understanding of EBV and NHL could lead to new approaches to prevent lymphoma, and new methods to diagnose, monitor, and treat lymphoma patients.
To address tumor diversity and their varied responses to chemotherapy, we require the ability to custom design treatment for each tumor. The goal of pharmacogenomics is to generate gene expression signatures for tissue using microarrays that are then correlated with prognosis or response to therapeutics. However, much of the complexity and diversity of response may be due to proteomic differences. Thus far, probing for proteomic diversity and linking this to therapeutic efficacy has been difficult. Our overall goals are to develop surface proteome signatures (SPS) and perform functional proteomic target validation analysis directly on primary tumor tissue. There is a great need to assess the efficacy of different chemotherapy combinations directly on patient tissue samples. A major difficulty is this respect is our inability to assess the small amounts of primary tumor tissue available from patient-derived samples. As part of our previous IMAT-funded research, we developed a library of single chain (scFv) phage display antibodies that recognize approximately 500 components of the surface proteome. In this work we also developed high-throughput methods for immunocytochemistry (ICC) using scFvs and apoptosis assays that use small number of cells (< 500) per assay. Together, these developments allow us to generate SPS and measure apoptosis after chemotherapy treatment using small amounts of primary tumor tissue. Cells will be tested with a battery of drugs alone and in combination and analyzed for apoptosis. Thus, a differential response to therapeutics will be correlated with a SPS. We will develop and test these assays in the R21 phase using thymic lymphoma mouse cell lines derived from three mouse models: transgenic MyrAkt and two genetic deletions, PTEN-/+ or p53-/-. This is an ideal model system, as the primary tumors are genetically defined by single oncogenic mutations. We will establish SPS for cell lines derived from thymic lymphoma lines from these mice. We will test for the efficacy of different drug regimens to obtain the optimum combination for inducing apoptosis of the cells. We will then test these drug combinations in primary tumor tissue from MyrAkt mice. We will also address the causal link between SPS and drug response and will test the functional role of scFvs that are biomarker candidates. In the R33 phase we will test the predicted optimal drug regimen on thymic lymphomas in the three mouse models, examining tumor load and survival. We will also characterize ten of the scFvs as potential biomarkers or targets for drug discovery. Our studies complement pharmacogenomics and provide a novel route to pharmacoproteomics.
R21/R33 CA107785-02 2004 MAYER, BRUCE J UNIVERSITY OF CONNECTICUT SCHOOL OF MEDICINE & DENTISTRY A high- multiplexed phosphotyrosine profiling assay
Protein tyrosine phosphorylation plays an important role in many of the biological processes involved in tumorigenesis, progression, and metastasis, and thus the global pattern of tyrosine phosphorylation of a tumor cell is highly relevant to its biological activity. It is likely, therefore, that molecular diagnostic methods based on the detection and characterization of tyrosine phosphorylation patterns in tumors will be useful for classification and prognosis. In the cell, most tyrosine phosphorylated sites on proteins bind tightly and specifically to small modular protein binding domains, in particular Src Homology 2 (SH2) domains. We have recently described a method, termed SH2 profiling, in which a battery of SH2 domain probes is used to profile the global state of tyrosine phosphorylation of a protein sample. In the current proposal, we will develop a novel multiplexed SH2 profiling format based on the labeling of SH2 domain probes with unique oligonucleotide tags. This novel approach will result in a quantitative profiling assay that is rapid, robust, reproducible, and sensitive enough for routine analysis of clinical specimens. In the R21 phase of the proposal we will demonstrate the feasibility of the oligonucleotide-tagged multiplexed (OTM) method and also develop two other quantitative SH2 profiling formats that can be used to validate the OTM method and to establish the utility of the approach for classification of tumor samples. In the R33 phase we will further develop and optimize the OTM method to incorporate the entire complement of approximately 150 phosphotyrosine-binding modules in the human genome and evaluate the performance and cost-effectiveness of different methods of quantitation. We will also fully develop bioinformatic tools to analyze quantitative SH2 binding data and cluster samples based on similarities in binding patterns and perform pilot studies on clinical samples to assess the usefulness of such data for molecular diagnostic classification of cancer. These studies will provide a novel tool for classifying tumors based on tyrosine phosphorylation patterns, which is likely to be useful in predicting the course of disease and response to therapy. They will also establish the feasibility of the OTM approach as a more general proteomic tool for the rapid and sensitive profiling of clinical samples.
A large number of transcription factors have been impliacted in tumorigenesis, yet little is known about their genomic binding sites under normal and pathological conditions. In order to understand the molecular mechanisms whereby abnormal functions of these factors lead to cancer, we propose to develop a genome wide location analysis (GWLA) technique that allows the rapid identification of direct in vivo targets for transcription factors. This approach involves formaldehyde fixation of cells, immunoprecipitation of crosslinked chromatin DNA fragments, and detection of enriched transcription factor binding sites with DNA microarray technologies. The GWLA technique has several distinct advantages over existing ones: First, the method directly examines the in vivo protein-DNA interactions throughout the genome, and can reveal functions of a transcription factor under both normal and diseased states. Second, this unbiased approach : does not require prior knowledge of a transcription factor's function, therefore can uncover its never biological properties. Third, the method has broad applications and can also be applied to discovery of DNA methylation patterns or mapping of other functional elements in the genome relevant to tumorigenesis. Through extensive preliminary experiments, we have verified the utility and exquisite sensitivity of this method with many transcription factors in both yeast and human cells. In the R21 phase studies, we will develop quantitative measures to assess the robustness and reliability of this method. In addition, we will demonstrate that this method can be used to map transcription factor binding sites in mouse genome, in the R33 phase, we will further develop and fully implement a GWLA system to identify targets for human and mouse transcription factors. Because a vast majority of known transcription factors bind close to gene promoters, our GWLA system will be focused on examination of promoter occupancy by specific : transcription factors in cells. We will first annotate gene promoters in the human or mouse genome, and then build DNA microarrays to represent these regions. We will also establish a standard protocol for target identification, and validate the performance of our system using a number of cancer-related transcription factors. This system should prove to be a powerful tool in mechanistic studies as welt as cancer diagnosis.
R21/R33 CA105514-02 2004 SIMS, CHRISTOPHER E UNIVERSITY OF CALIFORNIA IRVINE Assaying Tyrosine Kinase Activity: A New Paradigm
New approaches are needed in the molecular analyses of the growth-promoting signal transduction pathways involved in the development and maintenance of cancer. The Laser Micropipet System (LMS), a new technology for the biochemical assay of kinases, holds the promise of directly assaying the activities of protein tyrosine kinases in primary patient cells. The LMS was developed for the assay of serine/threonine kinases in individual cells grown in tissue culture. This R21/R33 application proposes to expand the applications of this instrumentation to directly determine the activity of oncogenic tyrosine kinases in primary cells from patients. Assay of Bcr-Abl in chronic myelogenous leukemia (CML) will demonstrate the power of this tool for research, clinical, and pharmacologic applications. While this assay will be of value in the study of the biology and resistance mechanisms in CML, the studies will lay the groundwork for a new paradigm in the study of molecular mechanisms in cancer signal transduction and will have general applicability. The R21 phase of this proposal is designed to establish the feasibility of assaying Bcr-Abl activity and to demonstrate the necessary assay conditions for measurements in cells derived from a patient with CML. The specific aims are to: 1) identify and characterize a reporter of intracellular Bcr-Ab1 kinase activity, 2) define and optimize conditions for measuring Bcr-Ab1 activity in human cells, 3) compare reporter phosphorylation in cells expressing Bcr-Ab1 vs. cells lacking this kinase, and 4) demonstrate the feasibility of using primary patient cells in kinase assays. The goal of the R33 phase is to demonstrate the potential for the LMS-based kinase assay in applications for the study of the biology of pharmacologic resistance in CML, including from primary patient cells. The R33 specific aims are to: 1) develop a membrane-permeant Bcr-Ab1 kinase reporter, 2) demonstrate the ability to detect imatinib resistance in CML cell lines, 3) determine Bcr-Ab1 activity in primary cells from patients with CML and their response to pharmacologic inhibition, and 4) determine if the assay can detect the emergence of imatinib resistance in CML patients and can differentiate the mechanisms of drug resistance.
R21/R33 CA109872-02 2004 WANG, YUE VIRGINIA POLYTECHNIC INSTITUTE AND STATE UNIVERSITY Comprehensive Analysis of Microarray Gene Expression
The challenge of cancer treatment has been to target specific therapies to pathogenetically distinct tumor subtypes to maximize efficacy and minimize toxicity. However, tumors with similar histopathological appearance can follow significant different clinical courses and show different responses to therapy. The recent development of gene microarrays provides an opportunity to take a genome-wide approach to predict clinical heterogeneity in cancer treatment. Although such global views are likely to reveal previously unrecognized patterns of gene regulation and generate new hypotheses warranting further study, widespread use of microarray profiling methods is limited by the need for further technology developments, particularly comprehensive bioinformatics tools not previously included by the instruments. The long-term goal of the proposed work is to develop, test, and disseminate effective bioinformatics tools to interpret the rich information derived by gene microarrays about underlying cancer biology (e.g., molecular biomarkers) and to facilitate molecular classification/prediction of cancer and response to therapy. This technology-driven project is inspired by the underlying hypothesis that microarray based gene expression profiling and integrated intelligent bioinformatics tools can, at the molecular level, (1) confirm existing and discover previously unrecognized cancer phenotypes; (2) identify most relevant diagnostic or therapeutic biomarkers; and (3) predict diagnosis, prognosis, and response to therapy. The R21 project will focus on: (1) performing rigorous and quantitative tests to compare the proposed methods with comparable existing methods, and (2) performing quantitative tests to show the feasibility of the proposed methods where no comparable method exists. The R33 project will focus on: (1) establishing a database of gene expression profiles derived from two human cancers (breast & childhood tumors); (2) extracting and refining most relevant biomarkers associated with previously and newly defined cancer phenotypes; and (3) developing, optimizing, and validating neural network classifiers to predict tumor phenotype and response to therapy with confidence values. These novel bioinformatics tools will be developed based on state-of-the-art and/or latest inventions in engineering, computer science, advanced statistics, and neural networks, and will produce a major advance in the molecular analysis of cancer.
R21/R33 CA107943-02 2004 ZHAO, YINGMING UNIVERSITY OF TX SW MEDICAL CENTER-DALLAS A Novel Proteomics Technology for Protein Farnesylation
The identification and characterization of changes in the level of farnesylated proteins in response to the treatment of farnesyltransferase inhibitors (FTIs), a newly introduced family of antitumor agents currently undergoing clinical evaluation, represents a major scientific challenge. Extant proteomics methods are limited to quantifying a few thousands of the most abundant proteins and, therefore, are unsuitable for the targeted profiling of less abundant farnesylated expression. The major goal of this application is to develop and validate a powerful technology, Tagging via Azido Substrate (TAS), for the efficient isolation of farnesylated proteins. This technology will then be applied to the proteomics analysis of farnesylated proteins in physiologically relevant models. The TAS technology involves the introduction of a synthetic azide-modified farnesyl substrate, either farnesyl azide diphosphate (FPP-azide) or farnesyl azide alcohol (F-azide-OH), which replaces the natural substrate during cellular protein farnesylation. The resulting farnesyl-azide (F-azide)-modified proteins will be affinity-purified through an azide-specific conjugation reaction (Staudinger reaction) using a phosphine capture reagent linked to photo-cleavable beads, which can then be released by UV light-induced photo-cleavage. Since affinity purification relies on covalent bonding resulting from a specific conjugation reaction between an azide and phosphine capture reagent, other proteins without the F-azide modification can be effectively removed by thorough washing. Thus, the TAS technology will allow farnesylated proteins to be isolated with high yield, high specificity, and low contamination. The initial focus of the R21 portion of this proposal is on the development of TAS technology and its application to the isolation of farnesylated proteins. These studies will be extended subsequently to geranylgeranylated proteins. The R33 proposal will aim at applications of the TAS technology to the identification of novel FTI targets. These studies will provide fundamental information for the understanding of molecular mechanisms of FTI functions and are likely to identify novel targets for antitumor drug design.
R33 CA105295-01 2004 MUDDIMAN, DAVID C. MAYO CLINIC Ovarian Cancer Screening Using Comprehensive Proteomics
The development of biomarkers for earlier detection of ovarian cancer will greatly improve survival. Most women present with advanced stage disease with survival rates of roughly 20%. Advances in the field of proteomics now provide the tools necessary to effectively analyze the serum proteome, and the application of such technologies holds great promise for determining novel biomarkers. This proposal outlines methods to comprehensively discover, identify, quantify, and validate a panel of specific and sensitive ovarian cancer biomarkers for early detection. We propose the following Specific Aims: 1. Determine candidate ovarian cancer biomarkers defined by comprehensive biomarker analysis of serum. Emphasis will be placed on refining the most effective overall analytical strategy for comprehensive and reproducible measurements. Spectral peaks that most accurately distinguish cancer vs. control will be identified using high-resolving power technology. Biostatistics and bioinformatics will be used to prioritize candidate markers (according to specificity, sensitivity and predictive value) for further characterization. 2. Identification and absolute quantification of the candidate biomarkers. This will improve reliability and high-throughput capabilities in subsequent validation testing, and the identification of these proteins will contribute further to research into the biologic basis of the disease. We will use accurate mass measurements, tandem mass spectrometry, and non-redundant protein and genome databases applied to distinct selected controls. Furthermore, we will determine the range of values for both normal and cancer serum samples using stable isotope labeled internal standards. 3. Clinical validation of a panel of biomarkers in specific groups of women. Sera from defined cohorts of women will be used to accurately determine the sensitivity, specificity, and predictive values of individual and combinations of candidate markers in all aspects of disease (early/late stage, familial, remission and recurrence) and in large numbers of non-cancer controls.
R33 CA107844-01 2004 O'LEARY, TIMOTHY J. AMERICAN REGISTRY OF PATHOLOGY, INC. Recovery of Protein from Formalin-Fixed Tissues
One of the most promising approaches for understanding the molecular pathogenesis of cancer is to relate genetic changes, such as mutation or altered gene expression, to metastasis, treatment outcome, and survival, using high-throughput molecular biologic and proteomic methods. In cancers where the time between initial diagnosis and treatment and the appearance of metastases is long, clinical correlations must be obtained with formalin-fixed paraffin-embedded (FFPE) tissues. However, large-scale multiplex techniques, such as proteomic analysis, serial analysis of gene expression, and gene chip methods using FFPE tissue have previously proven unsuccessful. The long-term goal of our research program is to improve public health by using high-throughput proteomic and molecular biologic screening methods to identify the molecular and genetic signatures of cancer. The objective of this proposal is to employ tissue surrogates to identify the formaldehyde-induced chemical modifications to proteins that occur during normal histologic tissue processing and to develop methods to reverse these modifications. Tissue surrogates are highly concentrated solutions of proteins that form tissue-like plugs following formaldehyde-induced intermolecular crosslinking. These plugs can be histologically processed like normal tissue. Our central hypothesis is that formaldehyde adducts and cross-links formed during histologic tissue processing can be sequentially reversed by a carefully designed series of heating, dialysis, rehydration, and protein renaturation steps, carried out under appropriate solvation conditions. We have formulated this hypothesis on the basis of our strong preliminary data, which have shown that the reversal of formaldehyde-induced chemical changes to proteins is relatively facile in aqueous solutions but requires a different approach for tissue that has been dehydrated in the presence of organic solvents. The rationale for these studies is that their successful completion will provide a foundation for high-throughput proteomic screening of FFPE tissues. This will improve practical interventions for the diagnosis, treatment, and prevention of cancer and will facilitate the development of therapeutic agents. Our studies are innovative because we have developed a novel model system (tissue surrogates) ideally suited to identify the formaldehyde-induced modifications to proteins that occur during histologic processing. At the completion of this project, it is our expectation to have established a comprehensive understanding of the protein modifications that occur during tissue histology, together with methods for optimally reversing these modifications. This knowledge should result in the ability to carry out proteomic analysis using FFPE tissue.
R33 CA105405-01 2004 VIDAL, MARC DANA-FARBER CANCER INSTITUTE Integrated interactome mapping
Despite the considerable success of molecular biology to understand diseases such as cancer, many questions remain unanswered. One area that has remained particularly unexplored so far is related to the fact that since the majority of gene products mediate their function together with other gene products, biological processes should be considered as complex networks of interconnected components. It is increasingly apparent that such complex networks have properties of their own. Thus, to understand any normal biological process of interest, or any disease mechanism such as cancer, one should consider a "systems approach" that allows, in addition to precise studies of single proteins or RNAs one at-a-time, more global analyses of the properties and functions of the molecular networks in which they function (1, 2). Since many gene products mediate their function, and are regulated, through protein-protein interactions, we have proposed and demonstrated using the nematode C. elegans as a model system that one way to accelerate the discovery of candidate cancer genes and begin to assign their products to potential functional networks is to comprehensively map such protein-protein interactions to generate so-called "interactome" maps. We have also learned how to integrate such interactome maps with other functional genomic and/or proteomic maps. Such integration provides first-generation wiring diagrams of functional networks that include the worm orthologs of important human cancer genes. Here, we describe i) further applications of our newly developed technologies to improve the quality and reliability of our interactome maps and ii) the transfer of these technologies to the mouse proteome, a model more closely related to human cancer than C. elegans. Our specific aims are to: i) improve the quality and reliability of interactome maps by adapting a Multisite Gateway technology in order to systematically map discrete interaction domains, ii) improve the quality and reliability of interactome maps by using the versatility of the Gateway technology to exogenously express large numbers of proteins and thereby confirming protein-protein interactions, and iii) apply these technologies to the mapping of the mouse interactome that is more likely to be relevant to human cancer.
R33 CA097526-01A2 2004 ZANGAR, RICHARD C BATTELLE PACIFIC NORTHWEST LABORATORIES Proteomic Identification of NAF Biomarkers
Breast cancer is responsible for ~43,000 deaths per year, a number which could be reduced dramatically by early detection of the disease. Currently, detection of breast cancer relies primarily on physical examination and conventional mammography. While these procedures have improved early detection of breast cancer and thereby decreased mortality, they still result in relatively high rates of false-positive and false-negative diagnoses. In addition, breast cancer prognosis is based on histological examination and is inadequate for assessing micrometastases. Due to this inability to accurately predict the risk of recurrence, 50% or more of breast cancer patients are unnecessarily treated with adjuvant therapy. A more useful and accurate evaluation of breast cancer could be obtained if these current, observation-based methods were supplemented with a molecular assessment. A number of serum proteins are reported to be altered in individuals with breast cancer when compared with healthy individuals. There is not, however, any known circulating protein that is suitable to define the stage of breast cancer or as a general marker for breast cancer, especially in the early stages of this disease. Mammary ductal cells are the cellular origin for 70% to 80% of breast cancer cases. Nipple aspirate fluid (NAF), which is obtained from non-lactating women, contains proteins secreted directly by these ductal cells. As such, we hypothesize that NAF is a concentrated and selective source of protein biomarkers for breast cancer. Proteomic approaches offer an unbiased way to evaluate NAF as a source of biomarkers and is sufficiently sensitive for small NAF volumes (10 to 50 (l). Therefore, the goals of this proposal are to characterize the proteome of NAF and to undertake preliminary analyses to identify potential protein biomarkers. To accomplish these goals, we propose to undertaken the following specific aims: Aim #1. Characterize the Proteome of Pooled NAFs Using Mass Spectrometry. Aim #2. Identify Potential Protein Biomarkers in Individual NAF Samples from Women with Early-Stage Breast Cancer using Isotopic Labeling and Mass Spectrometry. Upon completion of these aims, we will have provided the first detailed characterization of the NAF proteome and identified a subset of NAF proteins that are potential markers of breast disease.