Skip Navigation

Click on any project title for a more detailed description of the project. For more information about any of these awards (e.g., PI contact information or associated publications), please use the corresponding project number to search for information at the NIH Reporter website.

Current Year | 2012 | 2011 | 2010 | 2009 | 2008 | 2007 | 2006 | 2005 | 2004 | 2003 | 2002 | 2001 | 2000 | 1999

Show/Hide All

Project # Year
of Award
PI Name(s)
Institution Title
Abstract Text (Official)
Molecular dissection of gene expression pathways in cancer has revealed many new targets for cancer therapy. Those targets include the components of abnormal transcription machinery. Proteins involved in regulation of transcription attained high priority due to the convergence of many signal transduction pathways at the transcriptional level. New molecular therapies directed to transcriptional targets have significant advantages over traditional therapies due to a precision of their interference with target gene expression. Consequently, small molecule inhibitors, DNA binding polyamides, protein-binding oligonucleotide decoys, as well as small interfering RNAs and their combinations are being developed for cancer therapy. While rapid progress in molecular genetics and medicinal chemistry delivers new "attenuators" of gene expression, the technologies of early and non-invasive assessment of cancers that would be amenable to these therapies are currently lacking. In particular, imaging technologies that report directly on gene transcription in cancer cells are critically important for both cancer phenotyping and staging, as well as for evaluating new therapies. The goal of the proposed research is to optimize and characterize far-red fluorochrome labeled oligodeoxyribonucleotide molecular reporter probes (ODMRs) followed by the investigation of their transcription-factor reporting properties. Using NF-kB as a model cancer-relevant transcription factor, the following specific aims will be pursued: 1) Optimization of design, synthesis, and in vitro testing of transcriptional factor reporter probes (ODMR); 2) Investigation of the potential of ODMR probes in detecting active transcription factor in live cells.
R21 CA114157-01 2005 BROWN, KATHLYNN C UNIVERSITY OF TX SW MEDICAL CENTER-DALLAS A High Throughput Diagnostic Assay for Lung Cancer
Within the United States, 170,000 new cases of lung cancer are diagnosed per year. Over 60% of these patients will die within one year, making lung cancer the largest cancer killer of both men and women. The correct histopathological diagnosis of a tumor is critical in determining the appropriate treatment. However, precise classification of tumors remains a significant biomedical challenge. Furthermore, tumors of similar histology can have different clinical outcomes, stressing the need for more detailed molecular classifications. Generation of ligands specific to receptor(s) on a surface of a lung cancer cell will impact clinical issues including functional diagnosis. Our overall goal is to generate a panel of cell-specific molecules that could be used to classify tumor types and utilize these cancer specific reagents in a high throughput diagnostic assay. Using phage display technologies, my laboratory has developed platform methodologies to isolate peptides that bind to and mediate uptake into specific cell lines. We have identified cell-specific targeting peptides for 25 different cell types, including 4 lung cancer cell lines. The isolated peptides display remarkable cell specificities, even among similar cells, and are able to discriminate between normal and cancerous cells as well as different lung tumor cells. This high discriminating power suggests that peptides could be identified that selectively bind to different tumor types, even those with similar classifications. We propose to expand this panel of lung specific reagents by isolating cell targeting peptides for 4 different lung cancer lines and then utilize these peptides as diagnostic reagents. These peptides will be assayed for affinity and cell-specificity. We will remove the peptides from the phage backbone and synthesize peptide scaffolds that retain their affinity and cell-specificity. We will develop a high throughput fluorescent assay based on peptide binding that will allow for a more molecular classification of lung cancer samples. The assay can be multiplexed so that multiple binding events can be examined on a single sample. At the end of this pilot project we will have developed a novel diagnostic platform that can be expanded to clinical samples. Furthermore, the technologies developed here can be applied to other forms of cancer.
R21 CA114487-01 2005 CHIOCCA, E. ANTONIO OHIO STATE UNIVERSITY Imaging Transcriptional Activation of Gliomas
Differences in the expression of several genes between normal brain and its tumors (such as glioma) can provide information useful for malignant glioma diagnosis and therapy. Gliomas arise in the brain and are characterized by heterogeneous regions of necrosis, apoptosis, proliferation, invasion and angiogenesis. Drugs are being developed to target such phenotypes but it is unclear what imaging or molecular correlate will such drugs use to assay responses. Recently, serial analysis of gene expression (SAGE) data for malignant glioma has become available through efforts of the Cancer Genome Anatomy Project (CGAP) and several genes have been identified that are over expressed in glioma and not in normal brain. Some of these genes have been postulated to correlate with a particular glioma phenotype, such as invasion or angiogenesis. We propose a set of technologies useful to translate CGAP's knowledge into imaging the transcriptional activation of these genes and correlate such activation with the observed phenotypic heterogeneity in in vitro and in vivo models. We plan to combine the ability of our infectious bacterial artificial chromosome (iBAC) technology to rapidly isolate and deliver into cells large genomic fragments (up to 150 kb) with the ability of MRI and bioluminescence imaging to image gene expression. Specifically, the R21 phase this project proposes to: 1- Verify that large 5' flanking regions of one glioma expressed gene (for SPARC) transcriptionally activates the MRI-imaging gene ETR and luciferase, 2- Image the transcriptional activation of this region in in vitro models of glioma, 3- Image the transcriptional activation of this region in in vivo models of glioma. Transgenic mouse models have shown that large regions of 5' flanking area are best at providing complex tissue-specific and developmentally correct gene expression. A large 5' flanking region of SPARC will thus be cloned upstream of luciferase and/or ETR in the iBAC system. Imaging will be performed in in vitro and in vivo glioma models. The information obtained in this R21 will justify further grants (R33, R01) where the transcriptional activation of other genes can be imaged, thus providing a quantitative anatomic map of transcriptional activation in different heterogeneous regions of gliomas (invading, angiogenic, hypoxic, proliferating areas). This provides a baseline for assessing the effects of therapies (drugs, radiation) on these tumor phenotypes. The conceptual scheme presented herein will also be applicable to a variety of diseases for which gene profiling analyses are available.
R21 CA114489-01 2005 CLAFFEY, KEVIN P. UNIVERSITY OF CONNECTICUT SCHOOL OF MEDICINE & DENTISTRY Identification of Immune Selected Breast Cancer Antigens
Immune-dependent responses are selective in defining non-self or aberrant antigen presentation. Unfortunately, long-term antibody production to human cancer antigens is limited. In an innovative and novel approach, we have developed a method to utilize primary immune reactions in tumor draining lymph nodes to provide the means to define biologically active tumor antigens originating from breast cancers. This novel approach combines a series of steps to coordinate the construction of low complexity antibody cDNA libraries and protein production that are used to identify tumor antigens using sensitive antibody microscale "antigen-trap" assays followed by LC-MS/MS antigen identification. Tumor antigens identified can then be verified as potential tumor antigens using biochemical, immunological and molecular methodologies. The methodologies applied are medium throughput platform based designed to rapidly evaluate matched lymph node and tumor samples from the same patient. This project applies innovative technologies that demonstrate that: a) tumor draining lymph nodes are immuno-reactive to aberrant breast cancer antigens and produce antigen-dependent somatic hypermutation in proliferative B germinal centers, b) antigen binding domains of somatic hypermutated antibodies synthesized as recombinant VH and/or VHVl/Vk ScFv proteins can specifically recognize and identify breast cancer antigens, and c) antigens identified can be verified as diagnostic for breast cancer sub-phenotypes. This R21 application proposes to expand and refine our methodologies to: 1) determine the diversity and effectiveness of immune-selection of tumor antigens in a larger patient population, 2) expand our ability to produce antibody molecules with appropriate structure and antigen binding, and 3) develop methodologies to incorporate antibody proteins synthesized into highly sensitive assays that can screen primary cancer, histological material and/or biological fluids necessary to evaluate the potential of antigens as diagnostic biomarkers.
R21 CA107887-01A1 2005 ELLINGTON, ANDREW D UNIVERSITY OF TEXAS AUSTIN Transducing Tumor Cell Antigens to Amplicons
There are numerous methods for identifying and probing genomic markers in tumor cells, most of which are based on nucleic acid amplification technologies. However, in many instances protein markers are going to be of even greater utility in identifying and classifying tumor cells than genetic markers. To this end, it would be extremely useful to have methods by which protein (rather than nucleic acid) markers could be amplified. In this proposal we outline a series of novel methods for transducing tumor cell antigens to amplicons, which can in turn be sensitively detected using methods common to nucleic acid diagnostics. In particular, it has previously been shown that nucleic acid binding species (aptamers) selected from random sequence pools can specifically interact with a wide range of protein targets, including those relevant to tumor biology. Aptamers typically bind their cognate targets with dissociation constants in the nanomolar range and can readily discriminate between even closely related proteins. For these reasons, aptamers should prove useful for recognizing a wide range of protein markers associated with cellular transformation. We have previously developed automated methods for the selection of aptamers. We now propose to use such automated methods to target cell surface antigens of tumor cells. Selected anti-tumor aptamers will be adapted to a number of important diagnostic methods, including methods to label tumor cells and methods to transduce tumor protein antigens into nucleic acid amplification assays, via two novel methods, proximity ligation assays and rolling circle amplification.
R21 CA112418-01 2005 FRANK-KAMENETSKII, MAXIM D BOSTON UNIVERSITY Fluorescence in situ detection of short DNA sequences
A radically new approach for the fluorescence in situ detection of DNA is proposed, which makes it possible to detect short (about 20-bp-long) single-copy DNA sequences in metaphase chromosome spreads and in interphase nuclei under non-denaturing conditions. The method of fluorescence in situ detection of short sequences (FISDOSS) to be developed will be exceedingly specific because a circular probe will be assembled via ligation of synthetic oligonucleotides on short DNA sequences opened up by specially designed peptide nucleic acids (PNAs). A high sensitivity will be provided by an efficient contamination-immune isothermal method of signal amplification: rolling-circle amplification (RCA) of the assembled circular probes with incorporation of numerous fluorescently labeled nucleotides. All procedures will be performed directly on slides and the final detection of interphase nuclei and metaphase chromosomes will be done by standard techniques using a fluorescent microscope. In Phase I, proof-of-principle experiments will be performed on arbitrarily chosen sites unique for the human genome. The goal of Phase I is to demonstrate, after initial optimization, that the short specific sequences can be effectively and specifically detected within non-denatured metaphase human chromosomes. The method will be extended to parallel multiple detection of various unique sites in the human genome. To demonstrate that FISDOSS is applicable to detect genetic markers of cancer, 12 appropriate sites associated with chronic lymphocytic leukemia (CLL) will be tested. The implementation of the project will yield a convenient fluorescent in situ technique with a great potential for reliable and highly sensitive diagnosis of cancer on the DNA level.
R21 CA112220-01 2005 GASTON, SANDRA MARLENE BETH ISRAEL DEACONESS MEDICAL CENTER Tissue Print Micropeels for Molecular Profiling Cancer
Molecular profiling has emerged as an important strategy for identifying marker "signatures" associated with the biological changes that characterize specific cancers. To realize the full potential of the wealth of new biomarker information, it is essential to develop strategies for profiling human tissue and tumor specimens that are workable in a clinical setting. Clinical specimens are heterogeneous, and tissue heterogeneity is one of the major sources of complexity that must be addressed in the application of molecular profiling to the analysis of human cancers. We have developed a set of novel "tissue print" techniques that allow us to profile the molecular markers over extended areas of human tissue and tumor samples without damaging the specimen. We first applied our new tissue print techniques in the profiling of protein markers associated with capsular invasion in radical prostatectomy specimens. More recently, we have discovered that, during tissue print collection, we can peel a layer of cells off of the specimen and that this process does not cause detectable tissue damage (as determined by surgical pathologists), and thus does not interfere with routine clinical surgical pathology. We have also shown that the cells collected in the tissue print "micropeel" are adequate for PCR and quantitative rt-PCR analysis, allowing us to score multiple molecular markers and assemble the results in "tiling patterns" corresponding to the specimen surface. In the project outlined in this proposal, we will work closely with the research and development team at Qiagen Inc. to optimize the yield of mRNA and DNA from our tissue print micropeels collected from human prostate and breast tissue/tumors specimens. We will then develop a proof-of-principle pilot application of the tissue-print micropeel sampling technique for prostate needle biopsies, one of the classes of specimens that must be conserved intact for clinical diagnosis. Our long term goal is to utilize tissue print techniques in the clinical setting to simplify the process of obtaining an adequate representation of human cancers in biopsies and surgical specimens, and to develop protocols for this tissue sampling platform to support both proteomic analysis and PCR-based DNA and mRNA profiling techniques. In addition to facilitating basic and translational research, the tissue-printing platform can also be utilized as a tool for dedicated clinical applications, to provide "molecular sections" of extended areas of the specimen when the marker profile is itself of potential diagnostic importance.
R21 CA116365-01 2005 GREEN, ROLAND D NIMBLEGEN SYSTEMS, INC. Mapping Regulatory Pathways in Cancers
We propose to apply the emerging technology of Chromatin Immunoprecipitation on microarrays (ChIP chip) to identify the direct targets of transcription factors (TFs) known to be involved in breast and colon cancers. During the R21 phase of the project, we plan to identify these targets using a newly developed set of oligonucleotide microarrays that contain 15 million 50mer probes that tile through the non-repetitive sequence of the human genome at 100 bp resolution. We will validate that our ChIP chip protocols work with this new tiling array set. We will also develop and refine two new protocols, microarray reuse and 4-color hybridizations, that will allow economical use of this tiling array set so that it will be a more practical tool for research labs. In the R33 phase of this project, we will use this tiling array set to identify the direct targets of 9 TFs known to be involved in breast and colon cancers. Once we have collected the direct targets of these TFs, we will develop custom arrays that have probes that tile through all of the binding sites in the direct targets. We will use these TF focused screening arrays to study binding patterns in Icelandic breast and colon cancer samples to determine whether TF binding patterns could be a useful means of classifying tumors.
R21 CA112586-01 2005 GUDKOV, ANDREI V CLEVELAND CLINIC LERNER COLLEGE OF MEDICINE-CWRU ISSA: novel functional approach to cancer-related genes
Identification of genes and pathways that contribute to tumorigenesis should lead to the defining of novel targets for therapeutic intervention and provide biomarkers for better diagnosis, staging, and risk assessment for individual cancer patients. Further progress in molecular genetics of cancer would greatly benefit from a reliable methodology of assigning gene functions based on phenotypic changes resulting from modulations in gene expression. Existing techniques of this kind are based on screening of genetically modified cells for genetic elements favoring cell growth under restrictive conditions (positive selection). Recently, a novel gene discovery methodology, named selection subtraction approach (SSA), was developed that allowed for a direct negative selection. Although proven useful for isolation of killing or growth suppressive genetic elements, SSA's capabilities are limited by the necessity to construct expression libraries. This proposal is focused on developing a new Insertional Selection Subtraction Approach (ISSA) that combines the advantages of SSA with the power of retroviral insertional mutagenesis and is based on a completely new vector system. The insertional mutagenesis arm is enhanced by the addition of a regulatable promoter, splice donor sequences, and the ability to trap polyadenylation signals. The second arm of ISSA involves "tagging" or "bar-coding" each mutant (SSA), thereby allowing to monitor the relative abundance of mutants within the population during selection by using "retrophage arrays," the key component of SSA. ISSA technique promises to become a UNIVERSITYersal functional screening tool, free from major drawbacks of its precursors. After "technical" testing of ISSA, its power will be determined by identification of genes involved in regulation of cell sensitivity to TNF, a well-characterized system that has been already well studied by numerous approaches, including functional selection.
R21 CA112153-01 2005 HAAB, BRIAN B. VAN ANDEL RESEARCH INSTITUTE Longitudinal Cancer-Specific Serum Protein Signatures
Serum markers hold great promise for improving the care and treatment of cancer patients. Although many proteins have serum levels associated with various cancers, each has limited clinical usefulness when measured individually at single time points. The lack of sensitivity and specificity of current serum markers stems from heterogeneity in the baseline levels of the marker proteins and heterogeneity in the tumors and patients. A biomarker discovery strategy that accounts for the heterogeneity in people and tumors is to use individualized thresholds, based on longitudinal measurements, to precisely define abnormal levels for each individual. Previous research has shown that biomarkers defined by longitudinal measurements could have greatly improved specificity and sensitivity over current markers. No systematic study of this topic has been performed, largely because of the lack of a convenient technology for that purpose. A well developed and validated antibody microarray technology in the laboratory of Dr. Haab now makes this exploration possible. The multiplex detection capability of the antibody microarray will allow us to test the performance improvement upon using longitudinal measurements for many different proteins and to establish the general principles that define the use of longitudinal markers. In addition, the multiplex detection capability ultimately will allow the use of combined longitudinal measurements for even further biomarker performance improvement. To test this strategy, we will evaluate the sensitivity, specificity and time of the detection of prostate cancer recurrence using both longitudinal and single-time-point measurements of many different prostate cancer-related proteins in serum. This new approach addresses fundamental issues in biomarker research and should result in valuable information for a wide variety of research areas. The successful demonstration of the approach to prostate cancer diagnostics will signal its potential usefulness for all types of biomarker studies.
It is commonly accepted that the processes underlying the development of cancer are best modeled in vivo. A plethora of methods have been developed for genetic dissection of biological processes in somatic cells in culture; however, the adaptation of these techniques to in vivo use has been very limited. Our earlier experience indicates that reversible promoter-insertion mutagenesis offers a number of advantages over the currently used forward genetics techniques for cultured cells. The most important benefits are the dominant nature of the mutations, and the ease of identifying the affected gene and establishing the causal link between the mutation and the phenotype. The principal limitation for the adaptation of this technique to in vivo studies is the delivery of the insertional mutagen (typically, a retroviral vector) to the desired cell type. Based on our recent experience and published data from others, we propose a method to circumvent this limitation via the use of specially engineered transposon vectors. We propose to apply our technique to study the genetic events underlying melanoma development. We will screen for genetic events that cooperate with oncogenic Ras (commonly found in melanomas) in this process. We will confirm the properties of the proposed insertional mutagens in culture and then proceed to establish mouse strains suitable for mutagenesis. Upon verifying the properties of our constructs in vivo, we will selectively mutagenize melanocytes in transgenic animals and will test the functional link between tumorigenesis and the inserted promoter. Confirmed hits will be the subject of future investigation, while the technique itself will become available for dissection of various biological phenomena in general and aspects of tumorigenesis, in particular.
R21 CA114135-01 2005 KELLEY, SHANA O BOSTON COLLEGE Nanoscale Electrocatalytic Protein Detection
This proposal focuses on a novel system for the electrochemical detection of cancer related protein targets using a nanoscale electrode platform. The assay proposed relies on an electro catalytic process involving two transition-metal ions that reports on biomolecular complexation events. Because the reporter system responds to changes in the electrostatics of an electrode surface, it will enable the analysis of nucleic acids/protein and peptide/protein complexes. The proposed project has 3 specific aims: 1) electro catalytic detection of DNA repair proteins implicated in cancer using DNA-modified electrodes; 2) optimization of electro catalytic detection and multiplexed analysis of cellular DNA repair activities, and 3) development of a generalized electro catalytic protein detection method using prostate cancer biomarkers. The electro catalytic protein detection system described is advantageous because it will allow the analysis and discrimination of multiprotein complexes in addition to uncomplexed analytes and will have high selectivity and specificity. Additionally, the protein detection assays will be conducted using nanoelectrodes that will allow multiplexed detection of panels of different biomolecular targets.
R21 CA116141-01 2005 KRICHEVSKY, ANNA M. BRIGHAM AND WOMEN'S HOSPITAL Targeting miRNA in brain tumors.
MicroRNAs (miRNAs) are a recently discovered class of small 20-22 nucleotide non-coding RNA molecules that have been shown to regulate target gene expression in various organisms. By targeting the mRNA of protein-coding genes for either cleavage or repression of translation, miRNAs are thought to play critical roles in development, control of growth, proliferation, and cell lineage determination. However, the ability of this new class of regulatory RNAs to influence the processes of proliferation and differentiation in malignancy remains to be uncovered. Using an oligonucleotide array designed to detect the majority of mammalian miRNAs identifies thus far, we measured the expression levels of miRNAs in glioblastomas. This preliminary study identified a cluster of miRNAs that is up-regulated in glioblastomas. Among this cluster one miRNA is markedly elevated. Sequence-specific inhibition of this miRNA with modified antisense oligonucleotides induces apoptosis of glioblastoma cells in culture, suggesting a role for this miRNA in gliomagenesis. Similar regulatory miRNA molecules unique to tumor cells may exist and can serve as prognostic markers and, probably, excellent therapeutic targets for the treatment of high-grade brain tumors. The long-term goal of this proposal is to treat glioblastomas by targeting miRNAs. The immediate goal over the next two years is the development of technologies required for identification and validation of miRNA targets. We will create an oligonucleotide array for the complete profiling of miRNA expression in glioblastomas and characterize molecules expressed exclusively in these tumors. We will then develop ways to suppress these miRNAs in glioblastoma cell cultures and assess the effects on the migratory, apoptotic, and proliferative capacity of the tumor cells. Furthermore, we will test the potential of the miRNA inhibitors in animal models in vivo. Although beyond the scope of this proposal, in the ensuing years we will study downstream mRNA and protein targets that mediate miRNA functions. The results of the experiments proposed here will improve our understanding of biology of brain rumors. Moreover, miRNA targeting technology developed in this research could open the door to novel treatments of glioblastoma.
R21 CA114167-01 2005 LARSON, DALE N HARVARD UNIVERSITY Development of an Automated Frozen Sample Aliquotter
Biorepositories are a valuable resource in translational research for cancer and there are many such repositories in both academic and industrial settings. While these repositories are valuable, they are not immune from cost constraints and the approach to storing biological specimens (e.g., serum and plasma) involves a fundamental cost tradeoff between storing the samples in a larger number of vials each with volumes (100 ¨l to 400 ¨l) suitable for assaying or storing the samples in larger volumes (2 ml or 4 ml) to save freezer space. The first approach avoids downstream aliquotting and has only one freeze/thaw cycle but requires labor to aliquot the fresh sample and is volumetrically inefficient. The second approach requires aliquotting when the samples are requested and as a result the sample experiences a freeze/thaw cycle when it is processed for a study but is volumetrically efficient. A hybrid approach is also pursued where fresh samples are initially stored in a volumetrically efficient format until they are requested for a study and then returned to storage in the volumetrically inefficient format to avoid subsequent freeze/thaw cycles. This project will develop an automated instrument that will offer a new approach, combining the volumetrically efficient storage with the single freeze/thaw cycle. This instrument will extract aliquots from frozen samples without thawing the samples. Not only will this approach reduce the cost of operating these repositories while eliminating the second freeze/thaw cycle associated with the hybrid approach, but because it is automated it will increase the throughput in processing samples, reducing the 6 1/2 weeks to one week for a 1000 sample study, which is typical for the Nurses' Health Study. This team has demonstrated the ability to: (1) maintain the sample at -70 oC during processing; (2) extract aliquots from normal saline frozen at -80 oC, and (3) manage frost buildup during the procedure. The R21 project and the milestones have been developed to address the key technical risks associated with this instrument and the R33 project will deliver a prototype instrument that is suitable for use by the Nurses' Health Study to process samples for their collaborators. The design at this point will be documented well enough to enable its production in our lab in small quantities for use in other biorepositories.
R21 CA116079-01 2005 LIZARDI, PAUL M. YALE UNIVERSITY Global DNA methylation profiles of head and neck cancers
Head and neck cancer is a common disease worldwide, and more than 40,000 cases are diagnosed annually in the United States. The five-year survival rate is roughly 50% and has not improved over two decades. This research project focuses on the development of a new cancer biomarker paradigm based on the global epigenetic status of head and neck tissues. In many laboratories, DNA methylation is being examined as a means for early diagnosis of cancer. Recently DNA hypomethylation has been found to be a promising biomarker for the more advanced and aggressive stages of cancer, and there is an urgent need for analytical tools that will sample comprehensively the abnormal "methylome" hidden within the vast landscape of DNA repeats, in addition to the abnormal methylome of promoter-associated CpG islands. This grant application focuses on the validation of a novel microarray-based approach for epigenetic biomarker discovery and cancer classification that is complementary to other methods currently in use. The new method is relatively simple and has the unique capability to sample the methylation status of the majority of DNA repeats, as well as gene promoters, generating very large data sets. We will perform microarray-based methylation profiling using a statistically meaningful set of tumor samples from head and neck cancer patients. In addition, we will utilize the microarray-generated methylation profile information to identify clinically relevant subtypes of head and neck cancer, using a variety of analytical approaches. We will refine a subtype classification algorithm by inclusion of additional datasets available for the same samples, including allele gains and loses revealed by array-CGH and HPV infection status, as well as relevant patient clinical data. We will use this new information to address urgent diagnostic and prognostic clinical needs for improved classification of head and neck cancers in relation to diagnosis of early developmental stages, assessment of aggressiveness, likelihood of metastasis, and risk of recurrence after surgery.
R21 CA112149-01 2005 MELDRUM, DEIRDRE R. UNIVERSITY OF WASHINGTON Automated Cell Preparation in Tubes for 3D Microscopy
3D microscopy represents a powerful new cell analysis tool for early detection and diagnosis of cancer, but its future use may be limited because methods for preparation of samples are cumbersome, inefficient, labor intensive and generally imprecise. Current methods for cytological sample collection are manual and distributed in nature through various physicians' office laboratories and local hospitals, with the actual analysis being centralized at regional clinical laboratories. Among the cytological specimens are sputum, gynecological and colorectal scrapes, fine needle aspirates, urinary tract, and gastrointestinal samples. We propose the development of a new automated system that will transform these difficult and messy clinical specimens into an optimal format for 3D microscopy morphological and molecular analysis. The model and method we propose is comprised of three sequential steps. First, at the distributed site, an automated sample processor dissociates and fixes cells and debris for shipment in an automation compatible canister. Second, after shipment to a centralized clinical laboratory, the specimen canisters are loaded into an automatic processor that performs cleanup (debris removal), specimen/assay specific staining (and counterstaining), and finally embedding of cells of interest in glass microcapillary tubes (about 50 mu m ID), with cells being spaced at regular intervals (about 200 mu m) within a tube. This preparation format is uniquely suited for integration with multiple 3D imaging platforms for true 3D volumetric assessment of cell morphology and molecular probe and/or stain density distribution. The proposed system also enables use of cytometric flow sorting for enrichment of cells of interest at an intermediate stage of the sample preparation process. The potential impact to improved human health through rapid diagnostic screening will be illustrated using a high impact emerging technology, optical tomography. In summary, the aim of the proposed project is to develop, design, and build a complete sample processing system that automates the process of sample cleanup, assay specific staining, and mounting of cells into glass microcapillary tubes, and a tube positioning and rotation scanner mechanism for 3D microscopy analysis of cell morphology for the early detection of cancer.
R21 CA112228-01 2005 MURPHY, JOHN R. BOSTON MEDICAL CENTER Novel Targeted Reagents that Modify Oncogene Expression
A detailed understanding of the genetic basis of neoplastic diseases has emerged during the past two decades which has encouraged efforts to develop genetically targeted reagents both as experimental tools and as novel anti-cancer drugs. Peptide nucleic acid (PNA), a DNA mimic in which the phosphate deoxyribose backbone of DNA has been replaced by a pseudopeptide polymer, first described in 1991, has attracted particular interest as a gene-targeting reagent, since it is highly stable and binds to complementary RNA and DNA with high affinity and specificity. However, because PNA resists cellular uptake, its potential usefulness as a tool for modifying gene expression in whole animal studies or as a potential therapeutic agent has been limited. In preliminary studies, we have found that Anthrax toxin "protective antigen" (PA), the component of this microbial toxin that mediates cellular delivery, is able to transport ant/sense PNA oligomers into cells. To explore further the feasibility and potential of using Anthrax PA as a vehicle for delivering PNA into cells for the purpose of altering cancer-related gene expression, we propose studies with two specific aims. First, we will define the kinetics and dose limits of PA-mediated cellular delivery of antisense PNA, using PNA oligomers linked to varying polypeptide sequences derived from selected functional domains of toxin proteins. Stably transfected cell lines engineered to express a luciferase gene interrupted by a mutant beta-globin intron-2 (betaIVS2-654) with an aberrant splice site that can be blocked by antisense PNA, thereby allowing luciferase expression, will be used to detect antisense activity and effective cellular delivery of PNA. Second, we will determine whether Anthrax PA permits antisense PNA-peptide constructs to alter Bcl-x L gene expression and induce apoptosis in human cancer cell lines (e.g., PC3 cells) in vitro, and also to limit the growth and survival of these cells in vivo following implantation into nude mice. The potential impact of this research is substantial, not only with regard to the development of experimental tools for modulating cancer-related gene expression selectively and combinatorially in cancer cells in vitro and in vivo, but also with regard to the goal of developing genetically targeted agents for cancer treatment.
R21 CA116070-01 2005 PANNELL, LEWIS KENNETH UNIVERSITY OF SOUTH ALABAMA Automated Glyco-Analysis of Cancer Related Proteins
The quote that "aberrant glycosylation is the hallmark of cancer cells" is reflected in numerous reports in the literature documenting changes in glycosylation on specific membrane proteins in cancer cells relative to normal cells. These changes have been shown to be involved in the release of cancer cells into the extracellular matrix and in the formation of metastasis. Glycosylated proteins represent a huge, almost untapped source of biomarkers, considering the wealth of evidence documenting their significance in cancer. Unique glycoforms could be used for diagnostic purposes, to target drugs at cancer cells, and for the development of immunotherapy. Despite the evolution of new mass spectrometry based methods for protein analysis, few of these involve the determination of post-translational modifications, especially glycosylation. As routine methods (e.g., MS/MS based sequencing methods) yield little light on glycosylated peptides, this proteomics research facility has established a new approach to automatically identify glycan structures on pure proteins from commercial or recombinant sources. It involves the acquisition of molecular weight only spectra and the detection of the glycosylation patterns using accurately determined mass gaps between the various glycoforms. The presence of multiple glycoforms is used to enhance the analysis rather than to confuse it. The approach has been shown to be reliable and extremely fast (taking less than one second) at identifying and characterizing such sites, including in proteins with highly complex glycosylation patterns. The aim of this proposal is to prove its utility in cancer where changes in glycosylation changes are interlaced with the progression of the disease. It will concentrate on both the cell surface proteins and those secreted from cells. Data will be compared to previously published reports where available. The glycans on previously uncharacterized proteins will be established and validated against the best hand interpreted results. The long-term aim is to make glycoanalyses routine to all cancer investigators, and the software integral to the approach will be made publicly available on a www site. This will represent the first step, with the glyco-analysis of full proteomes being an ultimate objective.
R21 CA112145-01 2005 POOLE, LESLIE B WAKE FOREST UNIVERSITY HEALTH SCIENCES Profiling of Redox-Sensitive Signaling Proteins
Oxidative damage and redox signaling are important components of oncogenic cell transformation, yet the molecular details of how these phenomena impact cellular proteins remain largely uncharacterized. The goal of this proposal is to develop experimental and predictive methods for the identification and molecular analysis of proteins undergoing oxidative modifications at cysteine residues, impacting cancer-related redox signaling pathways in cells. With these efforts, we will be able to apply our new proteomics-based technology to the in situ identification of redox-sensitive signaling proteins in a multi-protein, multi-pathway, whole cell format for the first time. This will allow for investigations to proceed in both discovery- and hypothesis-driven modes to unravel the molecular details of cell signaling pathways responsive to the production of reactive oxygen species (ROS). Multiple studies have highlighted the importance of activated (low pKa) cysteinyl residues in proteins as the primary targets of oxidative modifications, and have shown that hydrogen peroxide is the most important ROS involved in receptor-stimulated cell signaling. The immediate product of peroxide-linked cysteine oxidation is cysteine sulfenic acid (Cys-SOH). Therefore, trapping of Cys-SOH upon its formation in cellular proteins using a detectable reagent will yield a sensitive and comprehensive way of locating redox-responsive cysteinyl residues in cell signaling pathways. To date, no methods exist to trap this species in a manner that is amenable to proteomics type approaches; thus, identification of Cys-SOH containing proteins must be done on an individual, targeted basis. Therefore, we propose four specific aims to (i) create and validate fluorophore- and biotin-linked reagents reactive toward Cys-SOH based on the known sulfenic acid reagent dimedone, (ii) develop "redox-profiling" protocols, using dimedone and reagents created in Aim 1, to trap Cys-SOH in proteins as formed within cells and detect the extent and location of Cys-SOH formation in proteins involved in a particular signaling pathway, (iii) develop "active site profiling" methods using bioinformatics approaches to identify particular cysteinyl residues likely to be targets of redox modifications, and (iv) validate and apply the experimental and predictive methodologies of Specific Aims 2 and 3 as tools in a total cell protein/proteomics format to analyze protein modification during the course of cancer-relevant cell signaling events. These tools will usher in a new era of redox proteomics and enable proteome-scale studies of effects of oxidative stress and antioxidant therapies on cell pathways and signaling networks.
R21 CA114143-01 2005 PUN, SUZIE H UNIVERSITY OF WASHINGTON Nanoparticles for efficient delivery to solid tumors
New technologies for molecular analysis of cancer identify patterns of genetic and protein expression changes that have occurred in tumorigenic cells. Application of these tools for in vivo analysis is critical for a complete understanding of metastatic cancer; sadly, such studies have been limited by the lack of effective methods for delivery to metastases. Nanoparticle formulations of these agents offer in vivo protection and concentrated tumor delivery and are therefore promising delivery entities. However, a major limitation of nanoparticles for tumor delivery is restricted interstitial transport. Here, we propose to harness forces generated by actin polymerization to propel nanoparticles within the interstitial space by energy-mediated, cell-to transfer, thus resulting in more efficient nanoparticle penetration. This goal can be achieved by realizing the following aims: (i) modifying nanoparticles with ActA, a bacterial protein that initiates actin polymerization resulting in propulsive forces, and optimizing formulations for motility in cytoplasmic extract, (ii) achieving actin-mediated, cell-to transfer of nanoparticles in cultured monolayer cells, and (iii) demonstrating improved nanoparticle penetration in three-dimensional spheroid cultures. Efficient delivery systems are crucial for both research and clinical applications; thus, successful completion of this project would result in a major step toward realizing the full potential of molecular analysis, detection, and treatment of cancer.
R21 CA116210-01 2005 SPENCER, FORREST A. JOHNS HOPKINS UNIVERSITY Genome Instability in Cells and Tissues of the Zebrafish
Change in genome structure can occur in mitotic and meiotic cell lineages, and this contributes to individual variation, evolution, and disease. It has been argued that stochastic somatic genome instability makes an early and important contribution to the development of human cancer, largely through loss of wild-type tumor suppressor genes. Many tumor suppressor genes themselves are guardians of genome structure and proper cell cycle control, and their loss may cause additional somatic instability. Thus, high levels of genomic instability may be viewed as possible cause and/or effect of steps in tumorigenesis. To distinguish these, a method is needed for continuous monitoring of genome stability in cell lineages that give rise to cancer. Genome stability in vertebrates is currently followed using karyotype analysis, fluorescence in situ hybridization, or measurement of endogenous marker loss using cell selection procedures. At this time, an in vivo system that can be used to determine genome stability in situ (i.e., without tissue disruption) is lacking. We propose the zebrafish Danio rerio as an ideal model system in which to develop this view of vertebrate biology, and we outline a novel method for following marker stability in fish. The method is based on a transcriptional repression design in which repressor loss leads to expression of the fluorescent protein EGFP. Using transgenic zebrafish, we will perform proof-of-concept tests for detection of repressor loss and characterization of repressor loss mechanisms. The zebrafish is ideally suited for development of this strategy due to the ease of organ visualization and its well-developed genetics and genomics tools. Furthermore, the rapid generation time and small size of the zebrafish supports cost-effective observation of many individuals, providing statistical power. In future work, this measurement of genome stability in situ will be important for understanding the relationship between gene function and genome instability in different tissues, and between genome instability and tumor development.
R21 CA113917-01 2005 WANG, BINGHE GEORGIA STATE UNIVERSITY Fluorescent Aptamers for Glycoprotein Detection
Early detection helps to increase the survival rate in cancer patients. One way to achieve this is the detection and analysis of molecular signatures or biomarkers that have been correlated to cancer development and prognosis. Along this line, there is a need for the development of new technologies for the molecular analysis of various cancer markers. Such spirit is reflected in an RFA (RFA-CA-05-002) requesting applications on developing new "detection technologies and sensors of cancer and the structures and molecules important in its development and diagnosis," among other things. In response to this RFA, we propose this feasibility study of a new platform technology that can be used for the rapid construction of fluorescent sensors for glycoproteins. We focus on glycoproteins because numerous such proteins have been implicated in cancer development. This method is based upon (1) the power of systematic evolution of ligands by exponential enrichment method (SELEX) in search of optimal oligonucleotide aptamers that can afford high affinity and specificity recognition of the target analytes, (2) the unique ability of boronic acids to recognize diol structures present on the saccharide part of glycoproteins, (3) our own development of several fluorescent boronic acid compounds that show very significant fluorescence intensity changes (17- to 200-fold) upon saccharide or glycoprotein binding. We hope to build synergy between the SELEX approach and the unique recognition of glycoprotein by boronic acids in making DNA aptamer-based fluorescent sensors that (1) have high affinity and specificity for the target glycoprotein and (2) exhibit very significant fluorescence intensity changes upon binding. Specifically, the project intends to develop a method to prepare DNA aptamers modified with our fluorescent boronic acid reporter compounds. The specific aims of the projects include (1) the synthesis of fluorescent boronic acid compounds that show great fluorescence changes upon binding to saccharides, (2) incorporation of the fluorescent boronic moieties into nucleotides, 3) using the SELEX approach for the selection of sensors with optimal specificity and affinity, 4) validation of the sensor binding with glycoproteins in solution. For this feasibility study (R21), we have selected prostate-specific antigen (PSA) as our model glycoprotein because of its importance in cancer diagnosis and the fact that glycosylation variations distinguish between physiological and pathological PSA isoforms. Such fluorescent sensors, if developed, offer the advantage of rapid and sensitive detection, the potential for high throughput screening, and low cost. Furthermore, the same technology, once developed, can also be used for the construction of fluorescent sensors for other cancer-related glycoproteins.
R21 CA111993-01 2005 WYMAN, ROBERT J YALE UNIVERSITY Genetic Methods for Detecting Gap Junction Communication
There is strong evidence that gap junctional communication (GJC) is a regulator of cell proliferation and that interruption of this is one of the steps in the malignant transformations of cancer. Gap junctions occur in all animal species and in most tissues from extremely early in development: the eight cell stage in mice, gastrulation in Drosophila and the two stage in nematodes. Yet gap junctions are the cell structures about which the least is known; their role in cell biology and development is still barely explored. Gap junctions are difficult to detect. The standard way to determine whether cells are GJ coupled is to inject dye into one cell and see if it spreads to neighboring cells. In vivo this requires microinjection, which limits the technique to large and unusually accessible cells. We propose to develop a molecular biological method for the in vivo detection of both enduring and transient GJC without the need for intracellular injection. In the simplest version of the technology, transgenic animals will be made with tissue-specific expression of b-galactosidase (b-gal). The intact animal will be injected with a b-gal substrate (e.g., X-gal) which is taken up by cells and is hydrolyzed to a small colored reporter molecule. b-Gal is too large to pass through gap junctions, but the reporter molecule can. Cells expressing b-gal can be detected with antibodies; any cell not expressing b-gal, but filled with the reporter color must have received its color via GJC. The technology will be validated for uniform cells of a single tissue type, different cell types in a complex tissue, in gap junctions made from a variety of GJ proteins, and in heterotypic junctions made from two different GJ proteins. Quantitative measures will be taken of in-animal and across-animal statistical reliability, extent of spread, and spatial and temporal resolution of the method. Aside from b-gal and X-gal as an enzyme-substrate pair, the method will be validated using other b-gal substrates. Another similar method will be tested using tissue specific expression of transporters to load the presynaptic cells and a detection method for trans-junctional passage. The method will be applied to tumors to assess GJ coupling between tumor cells, between tumor cells and normal cells before and after tumor induction. GJ proteins will be expressed in tumors and the method used to assess GJ coupling after expression and to determine whether tumor growth has been suppressed.
R21 CA114852-01 2005 ZHANG, HUI INSTITUTE FOR SYSTEMS BIOLOGY Profile serum proteins by glycopeptide capture and LC-MS
Cancers develop over a period of several years and they are characterized by molecular changes prior to invasion and metastasis. Development of a technology that enables screening of cancer from body fluids could permit cancer detection at early and treatable stages. It is expected that the composition of the serum proteome contains valuable information about the state of the human body in health and disease, and that this information can be extracted via quantitative proteomic measurements. Suitable proteomic techniques need to be sensitive, reproducible and robust, to detect potential biomarkers below the level of highly expressed proteins, to generate data sets that are comparable between experiments and laboratories, and have high throughput to support studies with sufficient statistical power. In this proposal, we will develop a method for high throughput quantitative analysis of serum proteins. It consists of the selective isolation of the peptides that are N-linked glycosylated in the intact protein using solid-phase extraction of glycopeptides (SPEG) on a robotic workstation, the analysis of these now de-glycosylated peptides by liquid chromatography mass spectrometry (LC-MS), and the comparative analysis of the resulting patterns. By focusing selectively on a few formerly N-linked glycopeptides per serum protein, the complexity of the analyte sample is significantly reduced, and the sensitivity, reproducibility, and throughput of serum proteome analysis are increased compared with the analysis of total tryptic peptides from unfractionated samples. We will explore the feasibility to identify cancer-specific serum proteins in the background of normal variation using a carcinogen-induced skin cancer mouse model. The specific aims are: 1) To develop chemistries and protocols for an automatic robotic system to isolate N-linked glycopeptides from serum in a high throughput and highly reproducible fashion; 2) To develop efficient and reproducible procedures for LC-MS analyses, and sequence identification of discriminatory peptides by tandem mass spectrometry; 3) To explore the feasibility of this method for the identification of distinctive serum peptides specific to cancer-bearing mice in the background of normal variations. If successful, the proposed research could subsequently be used for profiling human serum samples from cancer patients and normal individuals to identify the cancer-associated proteins in serum. The identified biomarkers will open a new paradigm for performing screening and detection of human cancer at early stage and for clinical therapeutic management.
R21/R33 CA112147-02 2005 HYSLOP, TERRY THOMAS JEFFERSON UNIVERSITY Nonlinear Models of mRNA Expression in Cancer by RT-PCR
The overall goal of this project is to improve the data analytic procedures associated with quantitative real-time polymerase chain reaction (Q-RT-PCR), with particular focus on measuring mRNA expression of molecular biomarkers and their association with colorectal cancer. Colorectal cancer is the third most common malignancy in the United States, and the third most common cause of death among cancer-related mortalities. While resection therapy is the main course of treatment for these patients, over 50% of patients presumed cured will recur within three to five years. It is hypothesized that these recurrences are actually undetected micrometastases. We propose to adapt Q-RT-PCR technology by changing the methods to quantify the amount of mRNA expression based on nonlinear models of the kinetic reaction obtained from Q-RT-PCR experiments. The current approach to quantitation does not make use of most of the data from the kinetic RT-PCR reaction, and the assumptions of the current model do not match the reality of the experiments. Thus, we also propose extensions of the error structure of these models to account for serial dilution of samples, heterogeneity across concentrations, and dependence of replicate samples. It is our hypothesis that improved measurement of the quantity of molecular biomarkers will offer more accurate prognostic information for colorectal cancer patients. Data from two NCI funded multi-center trials of guanylyl-cyclase-C (GCC) are available for these studies to demonstrate the clinical utility of our proposed methods.
Genomic, epigenetic and gene expression analysis from archived formalin-fixed paraffin embedded (FFPE) tissue samples with known clinical outcomes provides a unique opportunity for extraction of genetic information leading to improved cancer diagnosis, prognosis and therapy. However, extensive genotyping or microarray profiling on homogeneous cell populations within these samples often requires whole genome/mRNA amplification prior to screening. Major hurdles to this process are the introduction of amplification bias and the inhibitory effects of formalin fixation on DNA/RNA amplification. We have developed (RCA-RCA), a novel method based on isothermal rolling-circle amplification, that overcomes the limitations and promises to provide the needed link between obtaining a minute biopsy from partially degraded, FFPE samples and genotyping or micro-array screening. RCA-RCA enables whole genome/mRNA amplification that can be adjusted to the degree of FFPE sample degradation, as this is assessed via real time PCR. Thereby RCA-RCA enables retrieval of the maximum possible amount of information from the degraded sample. In the revised application, apart from adopting the Study Section's recommendations, a further enhancement of RCA-RCA is included, mRCA-RCA. mRCA-RCA amplifies DNA while retaining epigenetic modifications on a genome-wide basis ('whole methylome amplification'), thereby allowing highly expanded detection of methylation in fresh or FFPE samples. The R21 phase will examine the maximum capabilities of the technology and establish criteria for adjusting RCA-RCA to conform to the condition of the specific FFPE sample. The R33 phase will develop the technology for obtaining minute cancer biopsies from FFPE samples, assessing sample quality, and amplifying the whole genome/methylome (DNA) or transcriptome (RNA) without introducing amplification bias. Subsequently it will establish criteria and will validate the utility of the amplified material as input for the most frequently used molecular assays (mutation/SNP detection, microsatellite instability/LOH, array-CGH, expression profiling and methylation detection). By removing problems associated with sample degradation and biases associated with amplification this project will enable application of the newest technologies to the analysis of minute biopsies from archived tissue with known outcomes, thereby accelerating the process of candidate gene discovery.
R21/R33 CA112148-03 2005 OLIVI, ALESSANDRO JOHNS HOPKINS UNIVERSITY "Enhancement of Brain Tumor Immunotherapy by Fas-L RNAi"
Each year, approximately 185,000 people in the United States are diagnosed with a primary or metastatic brain tumor constituting the third leading cause of death in young adults ages 20-39. Among brain tumors, malignant gliomas are the most common and aggressive malignancies. Gliomas seem to be capable of inducing T apoptosis through the Fas/Fas-ligand (Fas-L) pathway. This mechanism allows them to circumvent immune surveillance by decreasing cell mediated immunity. Brain tumor therapy using immune modulators such as interleukins (IL) increases the recruitment of active T lymphocyte populations and has proven to be an effective strategy in experimental models of the disease. However, due to tumor-secreted Fas-L, the peritumoral Ts recruited by IL are activated through the trans-membrane Fas receptor, which initiates the caspase-3 mediated apoptotic cascade. Using RNA interference (RNAi) techniques, mRNA from tumor-derived Fas-L could be silenced and T cell apoptosis could be decreased, thus improving antitumor responses and potentiating the effect of interleukin therapy. RNAi sequences can be delivered by retroviruses, guaranteeing a constitutive transfection. In this proposal, the effect that treatment with Fas-L RNAi sequences delivered via retroviruses has on experimental gliomas will be investigated. The effect of this treatment modality will be studied alone and in combination with locally delivered IL incorporated into injectable microspheres. Treatment with Fas-L RNAi sequences is expected to decrease levels of tumor-derived Fas-L, therefore decreasing the rates of T cell apoptosis and increasing the populations of peritumoral T cells. Such an effect should allow a more consistent cell-mediated anti-tumor response able to prolong survival in animal models. Furthermore, the efficacy of locally delivered IL microspheres should be enhanced. The potential benefit of Fas-L RNAi delivered in this fashion could be applicable to several malignancies that have the Fas/Fas-L pathway among their immune privilege strategies.
R21/R33 CA111942-02 2005 TEMPST, PAUL SLOAN-KETTERING INSTITUTE FOR CANCER RESEARCH Peptide Profiling Techniques to Detect Thyroid Carcinoma
The information required for adequate diagnosis, treatment and monitoring of cancers is so complex that a panel of measurements, used in sum, may provide the best answers. The concept is embodied in SELDI-TOF mass spectrometric (MS) peptide profiling, an emerging technique for serum based cancer detection. Even though SELDI has thus far only produced low complexity spectra, the patterns, when analyzed as groups, have the potential to create learning algorithms with diagnostic accuracies as good as or better than conventional biomarkers. We have developed a system to capture peptides on magnetic reversed-phase beads, followed by MALDI-TOF MS, to yield increasingly complex, yet very reproducible patterns. This has clear advantages, as more displayed peptides provide more opportunity to select unique patterns ('barcodes') for cancer subtypes and stages, and to predict and monitor clinical outcome. Extreme care has also been taken to standardize specimen collection, handling and storage to avoid the introduction of artifact. Pilot projects at MSKCC with a variety of malignancies suggest that peptide patterns thus obtained appear to hold information that may have direct clinical utility. The goals of this project are to (i) automate our prototype serum peptide profiling platform and implement machine learning methods that use the resulting peptide patterns ('barcodes') for sample classification [R21]; and (ii) to test the 'barcode diagnostic' model in a high-throughput setting, using well defined and carefully observed groups of thyroid carcinoma patients [R33]. R21 aim one is to automate serum sample processing and analysis; aim two is to automate all data processing, to examine pattern selection and sample class prediction methods, and to integrate all software platforms; aim three is to develop routine MALDI-TOF/TOF tandem MS sequencing of 'barcode' peptides. R33 aim one is to define reproducibility of serum patterns in patients with thyroid disease; aim two is to determine barcodes that can distinguish patients with thyroid cancer from those with benign thyroid nodules; aim three is to assess if serum peptidome barcodes can identify occult metastasis in a large group of thyroid cancer survivors.
R21/R33 CA114149-02 2005 TUNG, CHING-HSUAN MASSACHUSETTS GENERAL HOSPITAL Imaging tumor associated fibroblast activation protein
The long-term goal of this research is to develop a novel fibroblast activation protein (FAP) sensing near infrared fluorescence reporter for early tumor detection and tumor classification. FAP is a cell surface antigen of reactive tumor stromal fibroblasts founded in more than 90% of epithelial carcinomas, but it is absent from epithelial carcinoma cells, normal fibroblasts, and other normal human tissue. Supporting tumor stromal fibroblasts are generally localized close to tumor vasculature, which is essential for early tumor development and growth. Thus it has been chosen as a target for monoclonal antibody based tumor therapy. FAP is not only a membrane protein but also a dipeptidyl peptidase. An imaging probe to report enzymatic activity and location of FAP could be extremely useful for early tumor detection. In this application we will develop a small molecular probe with ultra-sensitivity based on a unique class of fluorogenic chromophore that has significant changes in emission at different chemical states. Specifically, the probes emit no fluorescence in their initial intact state but become brightly fluorescent after specific proteolytic reaction. The newly developed low molecular weight, well defined fluorogenic probes are expected to have several advantages for imaging: a) fast tissue distribution allowing earlier imaging after injection, b) fast clearance allowing repeated imaging, and c) high likelihood of developing key candidates into clinically useful agents. The choice of FAP is based on its importance in tumor growth, invasion, and other processes in oncogenesis. Together with recent developments in fluorescence imaging technologies, this research is expected to ultimately result in clinical imaging agents with specificity for targeted enzymes. We believe that the developed approach can be used as a platform to design a broad spectrum of activatable molecular probes to image other amino peptidases in vivo.
R33 CA112141-01A1 2005 BAKER, JAMES R. UNIVERSITY OF MICHIGAN AT ANN ARBOR Photonic Crystal Fiber Probe Fluorescence Biosensing
Methods using fluorescent probes to identify cancer signatures and biological activities of cancer cells hold great promise. Probes based on fluorescence resonance energy transfer (FRET) and techniques such as fluorescence correlation spectroscopy (FCS) and other similar technologies offer the ability to identify specific RNA or protein molecules that can identify a cancer and provide information on oncogenic pathways used by the tumor cells. Other probes can give insight into drug response by measuring apoptosis induction by chemotherapies and radiation. However, fluorescent analysis has several limitations. Most ex vivo analyses use a flow cytometer or complex, confocal microscope to perform analyses, and this requires that tissue be removed from the body and often disrupted into cells, then fixed and analyzed in a static manner. The problems with in vivo fluorescent analysis are even greater since background fluorescence and tissue scattering, even in the near-infrared range, limit signal acquisition to the skin. Two-photon excitation has been a critical advance in optics, facilitating FRET, FCS and CARS techniques in vitro. However, these applications are limited by the complex technology (confocal microscopy) necessary to employ these techniques. We have demonstrated the use of two-photon fluorescence analysis through optical fibers for analysis of cancer cells in vitro and human tumors in vivo in SCID mice. This prior work constitutes the equivalent of an R21 proposal, as we achieved our major objectives: to develop sensing system optics and electronics and to document the ability of this system to obtain and analyze fluorescence signals in vitro and in vivo. The primary goal of this R33 application is to develop a more sensitive prototype device based on a novel dual-clad photonic crystal fiber (DCPCF) that we hypothesize will provide the sensitivity and redundancy necessary for the clinical evaluation of fluorescence signals in vivo using several fluorescence techniques. We plan to carry out our studies in three Specific Aims: 1: Develop DCPCF for use in a two-photon optical fiber fluorescence probe (D-TPOFF). 2: Utilize the D-TPOFF to quantify cancer signatures in vitro and monitor drug effects in tumor cells using targeted nanoparticles ex vivo and in vivo. 3: Utilize D-TPOFF to adapt other fluorescent techniques to examine events in tumors in vivo. At the end of these studies, this technology will be at a point where it is ready for commercialization.
Molecular dissection of abnormal regulation of cancer gene expression has revealed many potential targets for cancer therapy. Those targets include the components of normal, as well as abnormal transcription machinery. Proteins involved in regulation of transcription in cancer will attain high priority due to the convergence of many signal transduction pathways at the transcriptional level. New molecular therapies directed to transcriptional targets, including siRNA technology have significant advantages over traditional therapies due to a precision of their interference with target gene expression. While rapid progress in molecular genetics and medicinal chemistry delivers new +attenuators; of gene expression, there is a critical need in developing technologies that enable early and non-invasive assessment of cancer response to these therapies. In particular, enabling imaging technologies that report directly on gene transcription in cancer cells are critically important for both cancer phenotyping and staging, as well as for evaluating new therapies. Optical imaging in the near-infrared range of fluorescence combined with the use of enzyme-specific self- quenched probes has emerged as novel technology of live cancer cell screening. We previously devised a family of fluorescent probes based on synthetic biocompatible carriers of fluorochromes that report on hydrolytic activity in tumor-bearing animals. Recently, we developed new chemistry for generating asymmetrical as well symmetrical oligonucleotide molecular reporter probes (ODMR) designed to sense interactions with pleiotropic and evolutionally conserved components of transcriptional factor nuclear factor NFkappaB (NF-?B). NF-?B plays one of the key roles in tumor progression by regulating expression of cell adhesion, antiapoptotic and cytokine responsive genes in cancer and stromal cells in tumors. We recently tested novel synthetic approaches for introducing hydrophilic, non-interfering linkers into ?B-box sequences for covalent binding of fluorochromes to these probes to any internucleoside phosphate. We propose to develop ODMR technology that will be essential for further advancement of in vivo imaging of cancer-related target transcription activators.
R33 CA116123-01 2005 CHAURAND, PIERRE VANDERBILT UNIVERSITY MEDICAL CENTER Preparation of Cancer Tissues for MS Imaging of Proteins
Direct tissue profiling and imaging mass spectrometry (MS) provides a molecular assessment of numerous expressed proteins within a tissue sample. MALDI MS (matrix-assisted laser desorption ionization) analysis of thin tissue sections results in the visualization of 500-1000 individual protein signals in the molecular weight range from 2000 to over 200,000. These signals directly correlate with protein distribution within a specific region of the tissue sample. The systematic investigation of the section allows the construction of ion density maps, or specific molecular images, for virtually every signal detected in the analysis. Ultimately, hundreds of images, each at a specific molecular weight, may be obtained. To date, profiling and imaging MS has been applied to multiple diseased tissues, including human non-small cell lung tumors, gliomas, and breast tumors. Interrogation of the resulting complex MS data sets using modern biocomputational tools has resulted in identification of both disease-state and patient-prognosis specific protein patterns. These studies suggest that such proteomic information will become more and more important in assessing disease progression, prognosis and drug efficacy. Molecular histology has been known for some time and its value clear in the field of pathology. Imaging MS brings a new dimension of molecular information that specifically focuses on the disease phenotype. One important aspect of the MALDI MS imaging technology is sample preparation and processing. We propose here to further optimize the existing methodologies to maximize the information recovered from the MS analysis of fresh frozen sections, and develop and validate new approaches to investigate solvent fixed biopsies. Next, we propose to further develop and optimize methodologies to measure pharmaceutical compounds by MS in tissue sections. We also propose to further develop and validate protocols for the molecular analysis by MS of cancer cells in fine needle aspirates. Finally, we propose to automate some key aspects of these methodologies.
R33 CA112144-01 2005 D'ERRICO, FRANCESCO YALE UNIVERSITY Chemotherapy with injectable microdroplets
The aim of this project is to evaluate the usefulness of a novel drug delivery technology for cancer treatment, based on intravenously injectable emulsions of superheated perfluorocarbon droplets loaded with a therapeutic agent. The micron-size droplets dispersed in the emulsions are superheated, i.e., they are kept above their boiling point. In this thermodynamically metastable state, they can be vaporized by exposure to diagnostic levels of ultrasound, which permits the spatially and temporally controlled release of their drug content into a target region. The droplets are encapsulated with surfactants allowing them to withstand the mechanical stresses arising from inoculation and circulation in the bloodstream. Therefore they do not vaporize spontaneously, but only when triggered externally. The use of ultrasound as a triggering modality enables the integration of targeted delivery and imaging. The project is a collaboration between Yale UNIVERSITY, where the superheated emulsion technology was invented and drug delivery applications were proposed, and the UNIVERSITY of Michigan, where the use of superheated emulsions has been proposed and proven in vivo for occlusion therapy. The two groups, with complementary interests and expertise, propose to demonstrate the capabilities of the technology in a biologically relevant setting, i.e., the transport and release in vivo of paclitaxel, a highly cytotoxic chemotherapy agent virtually insoluble in water. The project will comprise investigations in various animal models available at our institutes to clarify different aspects of the delivery technology, as well as the utilization of various imaging techniques to confirm and quantify the occurrence and location of droplet activation. Specific aims of the project are: 1. To manufacture drug-loaded superheated emulsions and determine in-vitro and in-vivo, in dogs and rabbits, their viability for ultrasound-controlled drug-delivery; 2. To examine the acute effects on rats and evaluate the specificity of drug release and biodistribution in comparison with intravenous administration; 3. To use nuclear magnetic resonance techniques to measure and depict the distribution of bubbles in rats, and intravital microscopy techniques to observe directly the biophysical properties of blood-borne particles in the microcirculation of hamsters.
R33 CA112070-01A1 2005 FEDERSPIEL, MARK J MAYO CLINIC Technology to Optimize scFvs for Targeting Therapeutics
Antibodies provide superior targeting capabilities to a variety of therapeutic agents. Several technologies have greatly facilitated the initial identification of a variety of antibody reagents, including scFv and Fab antibodies, with virtually any possible specificity. However, lead antibodies often require further optimization to maximize their therapeutic performance: optimization of antibody expression and folding in relevant cells, and optimization of the affinity of the antibody for the target antigen. The development of promising targeting antibodies against cancer often languishes at this bottleneck. Therefore, technologies to facilitate antibody adaptation and optimization are urgently needed. Antibody optimization is best achieved by the randomization and subsequent selection of antibody mutants for the desired phenotypes since efficient rational design of antibodies is currently not feasible. Polypeptide display (e.g., phage display) is a powerful technology for the generation and screening of libraries of mutant polypeptides for a phenotype. A eukaryotic display technology that employs the efficient protein synthesis and quality control system of eukaryotic cells would best optimize the therapeutic parameters of targeting antibodies. We have recently demonstrated the feasibility of a retrovirus, avian leukosis virus (ALV), as a viral platform for the display of a variety of eukaryotic polypeptides including scFvs, and the efficient generation and selection of a peptide library in eukaryotic cells. The goal of this R33 application is to demonstrate the efficiency of using the ALV display technology for the optimization of the scFv scaffold for efficient folding and expression in eukaryotic cells and for generating a panel of scFvs with a range of affinities for their target antigen with an optimized scaffold. We will use the ALV display technology to optimize two scFvs with known specificity for tumor neovasculature: an anti-laminin scFv (L36) that inhibits angiogenesis in a variety of assays, presumably due to the exposure of laminin in the extracellular matrix during tumor neovessel formation; and a scFv that recognizes a VEGF:receptor complex (LL4) specific to endothelium in tumor neovessels. The ability of the nonoptimized and the optimized scFvs to target a therapeutic agent to tumor neovessels will be assessed using oncolytic measles viruses. Specifically, we aim to: 1. Create ALV display libraries of L36 and LL4 scFv mutants by error-prone PCR. 2. Screen the ALV display libraries of scFv mutants to generate a panel of L36 and LL4 scFv mutants with a range of known affinities (from ¨M to nM) for their target antigen and with an optimized scFv scaffold. 3. Generate recombinant measles viruses displaying nonoptimized and optimized targeting scFvs and compare them with respect to ease of production, efficiency of scFv display, particle to infectivity ratios, replication kinetics, and homing properties to tumor neovessels.
R33 CA114184-01 2005 FU, XIANG-DONG UNIVERSITY OF CALIFORNIA AT SAN DIEGO Typing the Transcriptome in Cancer Using Splicing Array
Alternative splicing is a permanent feature in higher eukaryotic cells and understanding of how alternative splicing alters the composition and function of the proteome represents a major challenge in the post-genome era. For cancer research, unique mRNA isoforms may provide a robust set of biomarkers for diagnosis and prognosis, and cancer-specific mRNA isoforms may serve as discriminating targets for effective therapeutic interventions. Furthermore, understanding of how splice choice is made and regulated in development and disease is a fundamental issue in cancer cell biology. An mRNA isoform-sensitive microarray technology would be ideally and timely suited for addressing a wide range of clinical and mechanistic questions regarding alternative splicing. In the past IMAT funding period, we have developed a unique and novel technology platform to attack the splicing problem. After a systematic and substantial effort in database construction and experimental development, the technology is now matured, and its superiority in reproducible measurement of mRNA isoforms under a variety of conditions has been demonstrated. Unique to the splicing array is the need to progressively enlarge the database for accurately annotated mRNA isoforms and preparation of corresponding oligo sets for measurement. We are therefore seeking IMAT support to put the technology in practical use and let the research community to take advantage of the technology development. We have three specific goals for the next phase in applying the emerging technology for molecular analysis of cancer. (1) We plan to use the technology to identify unique mRNA isoforms associated with prostate cancer. We will survey existing prostate cancer cell lines untreated or treated with androgen and estrogen as well as cancer tissues at different malignant stages to identify tumor-specific and hormonal regulated alternative splicing. (2) We propose to apply the technology to address mechanisms of splicing regulation by identifying direct targets for a large number of splicing regulators in knockdown and knockout cells. (3) Along with the proposed technology applications, we will progressively enlarge the high quantity databases coupled with the technology development and continue to improve and enlarge the database by adding new features and functions and develop linked software for splicing array data analysis.
Distortion of the cell genome characterizes neoplastic transformation. Genetic alterations that occur in tumor cells lead to activation of positive regulators of cell growth or survival and inactivation of factors that suppress these processes. A particular type of genomic alteration, chromosomal segment copy number imbalance, plays a significant role in malignant transformation: chromosomal deletions may inactivate tumor suppressor genes, while chromosomal segment amplifications may increase the gene dosage of oncogenes. In this study, we propose to apply a new technique, Comparative Hybridization of AP-PCR Arrays (CHAPA), which was developed in our laboratory, for high resolution profiling of breast tumors for DNA copy number alterations. This will allow the detection of single DNA copy number losses or gains at thousands of sites throughout the genome of the cancer cells (Specific Aim 1). We hypothesize that such genetic signatures may embrace the information on what cancer genes were responsible for the development and progression of each tumor and, consequently, the resulting pathologic behavior of tumor cells and their responsiveness to treatment. This general hypothesis will be tested by the analysis of genetic profiles to differentiate breast tumors according to their pathways of tumorigenesis (known or novel) and by the analysis of genetic profiles of breast tumors in association with their clinicopathologic characteristics, recurrence, and patient's survival to reveal genetic markers for cancer diagnosis and prognosis. Once frequent (common for independent tumors) genomic alterations have been identified, they will be compared with the loci known to play a role in breast cancer development. The genetic aberrations in chromosomal regions that do not contain known cancer genes will be selected for further characterization with the ultimate goal to identify the underlying novel cancer genes (Specific Aim 3). These experiments will provide a comprehensive view on the role of genetic aberrations in breast tumorigenesis. They will also help to identify genetic markers for breast cancer diagnosis, development, and prognosis and facilitate the identification, mapping, and eventual isolation of novel cancer genes.
R33 CA111933-01A1 2005 SCHWARTZ, DAVID C. UNIVERSITY OF WISCONSIN MADISON Single Molecule Genome Analysis of Oligodendroglioma
The proposed aims of this project center on constructing whole genome maps from 30 different oligodendroglioma tumor samples - a solid tumor that has confounded conventional genome analysis approaches to associate loss of heterozygosity (LOH) with a distinct set of gene(s). The research proposed reflects a multi-disciplinary collaborative effort to use a robust single molecule platform (Optical Mapping) to construct high-resolution restriction maps from a selected group of characterized tumors bearing a heterogeneous genome population. Chromosomal aberrations will be scored and classified on a whole genome basis in the absence of any hypothesis, outside of the established link between 1p/19q LOH and diagnostic purposes. Genomic aberrations in the tumor samples - deletions, insertions, translocations, tandem amplifications, and gross rearrangements - will be precisely located and characterized. Map coverage of 20-50x will ensure discernment of separate populations of chromosomal aberrations within each sample at 50 kb-500 kb genome intervals. New algorithms will be developed, based on Optical Mapping data, to identify breakpoints within a heterogeneous population of aberrant genomes based on the local alignment of single molecule barcodes, or Optical Maps, with the latest build of the human genome sequence. Aberrations will be statistically assessed to discern the percent of the tumor cell population bearing a given genomic lesion. To synergize this, a new generation of microfluidic device to incorporate cell lysis and DNA loading within the same disposable silicone fabrication will be perfected. These first-ever whole genome maps of oligodendroglioma tumor genomes and comprehensive determinations of aberrations will be entered into a customized Santa Cruz Genome Browser as additional annotation tracks. This technology provides a unique platform to decipher the complex molecular anatomy of cancer cells, on a whole genome basis, at high resolution.
R33 CA111940-01 2005 SHIBATA, DARRYL K UNIVERSITY OF SOUTHERN CALIFORNIA How Do Colorectal Cancers Arise Despite Surveillance?
Colorectal cancers are widely believed to develop through an adenoma-cancer sequence. By this paradigm, all cancers should be preventable with surveillance and polypectomy. However, in most surveillance studies, some cancers inevitably appear only a few years after negative clinical examinations. It is uncertain how such "interval" cancers appear, but either an adenoma was missed during the last "negative" examination, or there was an unexpected "rapid" mode of progression. A large number of such interval cancers have been found during an ongoing clinical surveillance program of high risk individuals with germline mutations in DNA mismatch repair genes (MMR), or hereditary nonpolyposis colorectal cancer (HNPCC). These interval cancers provide unique opportunities to rigorously understand why prevention fails. The key emerging technology is a new capability that infers time from cancer mutations. A molecular tumor clock can quantitatively infer times since MMR loss, and ages of final cancer expansions---the more mutations in a cancer, the greater these intervals. Distinguishing between failure from inadequate surveillance ("missed adenomas"), and failure due to the unique biology of HNPCC colorectal cancers ("rapid histologic or genetic progression"), is critical for the development of more effective strategies to prevent and treat colorectal cancer.