Harvey R. Herschman - US grants

My laboratory has utilized both genetic and molecular approaches to study the initiation of cellular proliferation. (1) 3T3 mutants unable to mount a mitogenic response to epidermal growth factor (EGF) and 3T3 mutants unable to proliferate in response to the mitogen/tumor promoter tetradecanoyl phorbol acetate (TPA) have been isolated and characterized. (2) Mutants resistant to the toxic action of EGF coupled to the A-chain of ricin toxin have been selected and characterized. (3) Seven cDNAs, called TIS (TPA Induced Sequences) genes, that are rapidly and transiently induced in response to TPA have modulators (TIS28/c-fos), secreted proteins (TIS7), and proteins of as yet unknown function (TIS10,TIS11, TIS21). The TIS genes can also be induced by other ligands, in a variety of cell-types. SUrprisingly, while no TIS gene is induced only by TPA, we have observed cell-type specific restriction of expression for several TIS genes. Our goals for the next five years include characterization of the functions of the TIS genes, as well as the range and basis of the cell-type restriction of their expression. FUnctional studies will include antibody localization of the TIS proteins, identification of genes whose expression is regulated by the DNA-binding TIS proteins TIS1 and TIS8, the consequences of overproduction and aberrant production of TIS proteins, and the consequences of inhibiting TIS gene expression, using antisense oligonucleotides somatic cell hybrids, transfected reporter genes fused to regulatory regions of the TIS genes, gel-retardation analyses, and chromatin structure probes. Our laboratory (and others) has emphasized isolation and characterization of mitogen-induced genes. However, cellular proliferation is also regulated by inhibitory factors. We will isolate and characterize both primary-response and secondary-response genes induced in response to the proliferation-suppressing protein TGFbeta. Our genetic approach to mitogenesis will continue. We will pursue the biochemical and molecular defects we have identified in our TPA nonproliferative 3T3 mutants. We will also isolate EGF + FGF "double mitogen" nonproliferative mutants, defective in essential post-receptor steps in the proliferative response to these polypeptide growth factors.

Diacylglycerols (DAGs) are found in the colon lumen from dietary sources, desquamated epithelial cells, and lyzed bacteria. They may serve as promoting agents for this cell type. A series of saturated and unsaturated DAGs will be assayed for biological activity parallel to that already known to be induced by TPA on 5 classes of human colonic epithelial cells in primary culture. These classes are different stages in the development of colon carcinomas. Specifically we will measure plasminogen-activator release from carcinomas; plasminogen-activator release from advanced stage premalignant cells from villous adenomas (benign tumors); the destruction aznd invasion of adenoma colonies by cocultivated carcinoma cells; the proliferative fraction of early stage premalignant cells from tubular adenomas; the proliferative fraction in cultures of very early stage preneoplastic cells from familial polyposis patients; the proliferative fraction and the steady-state levels of keratin polypeptides in normal cells. The ability of the biologically active DAGs to inhibit the binding of 3H-phorbol dibutyrate to colon carcinoma cells will be determined. Inhibition of the promoting effects of DAGs by the calcium soaps of monocarboxylic and dicarboxylic fatty acids, also found in the gut, will be measured. The effect of pretreatment of colonic epithelial cells with retinoids on DAG stimulated promotion will be assayed.

We are interested in (i) the molecular mechanisms by which stimulus- induced neuronal depolarization is converted into transcription-dependent changes in neuronal and behavioral properties, and (ii) the mechanisms by which neurotrophins specify neuronal differentiation of neuronal precursor cells. To study these mechanisms we are identifying and characterizing "neuron-restricted" immediate-early genes (IEGs) preferentially induced by depolarization, and genes that are induced by NGF, but not by other stimuli. We constructed a phage library prepared by subtracting cDNA from stimulated PC12 pheochromocytoma cells with cRNA from stimulated fibroblast and hepatoma cells. The library was differentially screened, to identify cDNAs for "neuron-restricted" immediate-early genes. One clone encodes the rat homologue of synaptotagmin 4 (Syt4), a recently identified member of the synaptotagmin family. Syt4 is induced in PC12 cells by depolarization, calcium ionophore, and secretogogues, but not by growth factors or tumor promoters. Syt4 is present in brain, but not in other organs. Syt4 is induced in the hippocampus and the piriform cortex following kainic acid induced seizures. In contrast, Syt1 is not inducible in PC12 cells or brain. We also identified secretogranin as a neuron- restricted IEG induced by depolarization, but not growth factors. In the next grant period we will compare the biochemical properties of Syt4 and Syt1 to determine (i) if Syt4 can, like Syt1, also form complexes with neurexin, (ii) if Syt4 is a vesicle protein, (iii) if Syt4 and Syt1 are present in overlapping or distinct vesicle populations, and (iii) if the rates of synthesis and degradation of Syt4 and Syt1 differ. We will determine whether altering Syt4 expression in PC12 cells modulates depolarization-induced fusion of synaptic vesicles with presynaptic plasma membranes and/or neurotransmitter release. We will also use genetic approaches to study Syt4. We will create mice with disruptions in the Syt4 gene, for electrophysiological and behavioral studies. We will map the marine Syt4 gene, to determine whether Syt4 might be a candidate gene for neurodegenerative or behavioral mutations. It is likely that additional depolarization-specific neuronal IEGs exist, but have not been identified. We will use Syt4 and secretogranin as guides to rescreen our subtracted library. We have modified the representational difference analysis (RDA) procedure to enrich for DNA sequences preferentially induced by NGF in PC12 cells, and identified collagenase as an NGF-inducible gene. We will use RDA to identify NGF-induced genes necessary for PC12 differentiation, and to identify additional depolarization-specific IEGs.

Prostaglandin production is a hallmark of inflammation in many tissues, including the airway (i.e. asthma) and gastrointestinal tract (inflammatory bowel disease). Prostaglandin secretion is modulated by cytokines, endotoxin and other inflammatory response mediators in macrophage, epithelial cells and fibroblasts. Until recently the mechanism of induced prostaglandin synthesis in the inflammatory response was unknown; although cytokine or endotoxin challenge increased synthesis of immunoprecipitable prostaglandin synthase (PGS), the mRNA for the known PGS (PGS-1; EC.1.14.99.1), was not coordinately induced. We have cloned a cDNA and gene, TIS10/PGS-2, for a second prostaglandin synthase. TIS10/PGS-2 message and protein are rapidly and transiently induced in macrophage, epithelial and fibroblast cells, in response to growth factors, cytokines, and endotoxin. Moreover, induction of both prostaglandin synthesis and TIS10/PGS-2 expression in response to inflammatory mediators and growth factors is blocked by glucocorticoids. Nitric oxide has recently been shown to also play a major role in mediating inflammatory responses. We have now demonstrated that the calcium-independent form of nitric oxide synthase (iNOS) is induced in many of the same cell as TIS10/PGS-2. We believe that interplay between prostaglandin and nitric oxide production, regulated by the induced expression of the TIS10/PGS-2 and iNOS genes, plays a critical role in immune inflammatory reactions in the respiratory and gastrointestinal tracts. In this project we will (i) characterize the iNOS gene, (ii) prepare antisera to the iNOS protein, (iii) determine the molecular mechanism(s) of INOS induction and its regulation by glucocorticoids and regulatory cytokines in macrophage, epithelial cells and fibroblasts, in comparison with similar studies with TIS10/PGS-2, and (iv) characterize the effect of polycyclic aromatic hydrocarbons on the expression of TIS10/PGS-2 and iNOS, in both the absence and presence of other inflammatory mediators, such as cytokines. We will also (v) create mice with homologous deletions of the iNOS gene, and characterize the inflammatory response phenotype of mice deleted for iNOS, for TIS10/PGS- 2, and for both genes.

DESCRIPTION (provided by applicant): During the current ICMIC cycle our goal has been to disseminate the principles and mind-set of noninvasive molecular imaging to the UCLA cancer research community, in keeping with the 1999 ICMIC RFA objectives. During the past four years, non-invasive imaging technology in small animal cancer research has permeated the campus. Fostered by the ICMIC, under the umbrella of the UCLA Cancer Center and the Crump Institute for Molecular Imaging, a campus-wide small animal imaging facility has been established. Two years ago we initiated an ICMIC policy to encourage application of molecular imaging technologies to translational studies with clinical endpoints. In the 2004 ICMIC RFA, the NCI has retargeted the ICMIC goals to "capitalize on the extraordinary opportunity for molecular imaging to have an impact on the diagnosis and treatment of cancer patients non-invasively and quantitatively." Research Component 1 of this application is a clinical extension of two of our previous Developmental Projects, to bring reporter gene marked T-cells to clinical trials to monitor TCL targeting to melanoma immunotherapy. Research Component 2 is also an expansion of a previous Developmental Project, to (i) monitor PET metabolic responses to targeted kinase therapies in the clinic and (ii) to develop, with culture and animal models, the optimal use of metabolic imaging to distinguish rapidly responders and non-responders. Research Component 3, an extension of a current Developmental Project, is creating a new non-invasive PET reporter gene mat will have minimal interfering effects and that makes use of positron-labeled reporter probes that are already in clinical trials. Research Component 4 extends to a pre-clinical metastasis model laboratory-based, proof-of principle experiments in monitoring adenovirus redirection and restriction to tumors. Our proposed Developmental Fund projects similarly emphasize translational studies to bring radio-nuclide based molecular imaging, through pre-clinical models, to clinical applications. Research Component projects, Developmental Fund projects and Career Development Award projects are supported by our three continuing Specialized Resources; (i) the Cytrotron and Radiochemistry Specialized Resource, (ii) the Molecular Imaging Specialized Resource and (iii) the Quantitative Image Analysis Specialized Resource. Our goals, through our Research Components, Developmental Fund and Career Development Component will be (i) to continue to foster the incorporation of non-invasive molecular imaging to the best of cancer research at UCLA and (ii) to recruit, encourage and provide resources to encourage translation of molecular imaging principles and practices to cancer diagnosis, management and therapy.

Prostaglandins modulate platelet aggregation, kidney function, immune responses, respiratory function, reproduction and other physiological processes. Abnormalities of prostaglandin biosynthesis occur in pain, fever, bone resorption, cardiovascular disease, inflammation and cancer. Cyclooxygenase/prostaglandin synthase (COX/PGS) catalyzes the synthesis of the common intermediate, PGH2, in the production of prostaglandins. We identified a second, inducible COX, COX-2. We also demonstrated that COX-2 induction is required for prostaglandin production in response to growth factors and inflammatory stimuli, a result that provides the rationale for the clinical utility of COX-2 specific inhibitors. Elevated COX-2 gene expression plays a role in many pathologies. The ability to measure COX-2 expression in living animals would permit us to (i) determine the role of COX-2 in inflammatory responses and tumor progression and (ii) evaluate the utility of monitoring COX-2 expression as an intermediary marker of both disease and therapeutic response. We developed methods to measure reporter gene expression repetitively, non-invasively and quantitatively in living animals. We express reporter genes whose protein products sequester positron-labeled probes, then detect reporter gene-dependent probe retention by using positron emission tomography (PET). We have developed two systems; (i) Herpes Simplex Virus 1 thymidine kinase (HSV1-tk) as the "PET reporter gene" and [18F]-8-acycloguanosines (ganciclovir, penciclovir) as the "PET reporter probes", and (ii) the dopamine D2 receptor (D2R) as the PET reporter gene and [18F] fluoroethylspiperone as the PET reporter probe. We validated these two PET reporter gene systems in both a somatic viral delivery model and in transplantable tumor models. We will develop procedures to quantitatively and non-invasively monitor PET reporter gene expression from COX-2 promoter-driven transgenes and from the endogenous COX-2 gene. We will create transgenic and "knock-in" mice in which the HSV1-tk and D2R PET reporter probes to monitor COX-2 gene expression in four inflammation models (the carageenan-injected paw, air-pouch inflammation and liver inflammation) and in two tumor induction systems [multi-stage initiation-promotion- progression skin carcinogenesis and intestinal neoplasia in min1(+/- )mice]. These mice will permit investigators to monitor COX-2 expression in a variety of paradigms.

ABSTRACT NOT PROVIDED

An integrated microarraying facility will be initiated in the Department of Molecular and Medical Pharmacology (DMMP) in the Center for Health Sciences (CHS) at UCLA. The facility will consist of a DNA processing robot, a microarrayer, a scanner, a DNA/clone resource, and a computational facility. The microarray facility has been designed to provide the highest practicable throughput of this powerful technology.

The research will cover the whole range of modern biology including studies of retroviruses, differentiation of neural stem cells and other cells, the cellular response to metals, synaptic transmission, neural growth factors, nitrous oxide physiology, immune system differentiation, neural aspects of behavior and glucocorticoid signaling. An important part is to improve the bioinformatics of microarray technologies using the data provided by the facility.

This facility will provide researchers and their students the ability to visualize gene expression patterns for thousands of genes at once and have a major impact on research in cell and molecular biology at UCLA.

DESCRIPTION (From the Applicant's Abstract): We have two goals in our neurobiological studies. Our first goal has been to identify, in neurons, depolarization-induced immediate-early genes (IEGs) that play a role in synaptic plasticity and to characterize the biochemical mechanisms by which these IEGs mediate synaptic function. Our second goal has been to identify, in neuronal precursors, IEGs induced preferentially by Nerve Growth Factor (NGF) versus other stimulating ligands and to characterize the biochemical mechanisms by which these NGF-induced IEGs mediate NGF-driven neuronal differentiation. We identified synaptotagmin IV (Syt IV) as an IEG induced by depolarization in the hippocampus, prepared anti-Syt IV antibodies and showed that Syt IV is a labile protein incorporated into synaptic vesicles after induced synthesis, where it can form oligomers with Syt I. We created a Syt IV knock-out mouse, and demonstrated that Syt IV (-/-) mice are deficient in acquiring two hippocampal-dependent learning tasks, but not in acquiring two amygdala-associated learning tasks. We subsequently identified four protein kinases (KID-l, PIM-1, SIK, MAPKAP-2) and two transcription factors (nurr1, rTLE3) induced by depolarization. These genes are additional candidate IEGs whose products may mediate synaptic plasticity. We also identified six messages preferentially induced by NGF (versus Epidermal Growth Factor) in the PC12 cell model system of neuronal differentiation. We demonstrated that one of these IEGs, the urokinase plasminogen activator receptor (UPAR), is required for NGF-driven PC12 cell morphological differentiation and secondary response gene expression, using UPAR antisense oligonucleotides and anti-UPAR antibody. We will determine the biochemical basis for Syt IV modulation of synaptic function and collaborate on studies of electrophysiological correlates of the Syt IV (-/-) behavioral deficits. We will determine if Syt IV and our depolarization-induced IEGs are induced via the CREB pathway, and whether they modulate depolarization-induced, calcium-dependent exocytosis. For those IEGs that appear to be good candidates for modulators of synaptic function, we will prepare knock-out mice and analyze their behavioral characteristics. To determine the role of UPAR in NGF-driven neuronal maturation, we will examine development of sympathetic and sensory ganglia in UPAR null mice. We will also use cultured peripheral ganglion cells from BAX/NGF null mice to characterize the role of UPAR in NGF-driven differentiation. Finally, we will determine mechanisms by which (i) NGF induces UPAR and (ii) UPAR modulates NGF-driven neuron differentiation.

DESCRIPTION: (provided by applicant) We seek renewal of the "Research Training in Pharmaceutical Sciences" (RTPS Predoctoral Training Grant). RTPS is designed for students who want to extend their knowledge of molecular and cell biology to include principles of modern pharmacology and molecular imaging, and the application of these disciplines at cellular and organismal levels. RTPS adds formal instruction in chemical, cellular and organismal pharmacology and in molecular imaging, and emphasizes their applications in both cellular and whole animal contexts. RTPS faculty have expertise in cell/molecular biology, biochemistry, pharmacology, genomics and molecular imaging. Development of microPET imaging procedures and PET reporter gene technologies now permit application of molecular and cellular pharmacology to living animals, whose genomes can be experimentally manipulated, to complement existing molecular and light microscopic techniques. RTPS provides vertical integration of research in molecular and cellular pharmacology from molecule to mouse to man, combining concepts and applications from molecular structure through molecular medicine. Proposed training faculty increase from 24 in the current period to 36 in this application, include 10 Professors, 12 Associate Professors and 14 Assistant Professors, and come from seven departments. A majority, 22, of the mentors have primary or joint appointments in the Department of Molecular and Medical Pharmacology (DMMP). DMMP dominance results from two factors: (1) DMMP emphasis on modern pharmacologic and imaging techniques and the interface of cell biology, pharmacology and molecular imaging in DMMP and (2) recruitment of 10 new faculty, with research interests at the heart of the RTPS program, into DMMP in the last five years. The large number of junior faculty mentors reflects an institutional commitment to recruitment of faculty with research interests that reflect RTPS goals. Students are selected for RTPS after their first year in graduate school, and are drawn from a pool that comes from individual degree granting programs, the UCLA ACCESS graduate admissions program, the MSTP program and the UCLA STAR program. RTPS began in 1998, with the appointment of two students. Because of an increase in interest in this area of research, both by faculty and by students, we are requesting four trainee slots per year. Resources of the DMMP and the Crump Institute for Molecular Imaging will be available to all RTPS students, regardless of the department in which they receive their Ph.D.

Since this submission is a competitive renewal, we will first present a brief Background to describe how our Developmental Funds have been/will be used. We support four Developmental Fund Projects annually, at $45-50K per project. Projects are for up to two years;second year funding competes in the pool with first year funding applicafions. Our initial goal for our Developmental Funds was to facilitate introduction of molecular imaging to UCLA cancer research;to bring the strongest molecular, cellular and cancer researchers into the imaging arena and to proselytize for use of non-invasive imaging in fundamental cancer research. In our current funding cycle, three of the four Research component Projects were new, and grew out of Developmental Funds Projects. Funding our competing ICMIC renewal is perhaps the best objective testimony that we achieved our goal. The narratives below describe proposed Developmental Fund Projects for year 1 of the renewal application. During this current cycle, the Jonsson Comprehensive Cancer Center and the Dean for Research for the David Geffen School of Medicine have also contributed funds to support related projects, in consultafion with Dr. Herschman (for details see Secfion A, Description).

D.I.I. Introduction: The Cyclotron and Radiochemistry facility is a Specialized Resource component of both the UCLA-ICMIC and the UCLA Department of Energy Molecular Medicine program. Our history with the DoE program predates the initiation of the NCI ICMIC, extending back for over three decades. The prime objective of the Cyclotron and Radiochemistry Specialized Resource is to foster all radiopharmaceutical needs of UCLA-ICMIC and UCLA-DoE investigators, and to serve other UCLA research projects that complement these research programs.

Adenovirus vectors are among the most popular vehicles for cancer gene therapy protocols. However, adenovirus infect cells via the Coxsackie and Adenovirus Receptor, which is often more extensively expressed on normal cells then on tumors. We utilized bioluminescent imaging following systemic Ad.CMVfLuc administration to monitor adenovirus transductional "untargeting" and "retargeting" by sCAREGF. sCAR-EGF is a bi-specific retargeting molecule containing the soluble portion of CAR linked to epidermal growth factor. Noninvasive optical imaging demonstrates that systemically injected [Ad.CMVfLuc] [sCAR-EGF] complexes have reduced ability to infect liver, but can infect EGF receptor-positive xenografts. COX-2 is not expressed in most normal tissues, but is overexpressed in many tumors. We used noninvasive optical imaging to examine fLuc expression following intratumoral injection of Ad.COX2fLuc (an adenovirus expressing luciferase from the COX-2 promoter). No expression occurs in liver. In contrast, expression occurs in COX-2 positive tumors. Colorectal cancer (CRC) is a leading cause of cancer death. CRC frequently metastasizes to liver. CRC liver metastases often over-express both carcinoembryonic antigen (CEA) and COX-2. We will combine our experience - both with transductional "untargeting" and "retargeting", and with restricted COX-2 gene expression - to develop effective therapies for CRC hepatic metastases. We will establish xenograft CRC models of liver metastasis. We will create Ad.COX2.fLucTK.COX2, an adenovirus that expresses a fusion protein containing both firefly luciferase and HSV1-thymidine kinase (fLucTK). CRC tumor-restricted fLucTK expression will result from regulation by both the COX-2 promoter and the COX-2 mRNA 3' untranslated region. Transductional liver untargeting and Ad.COX2.fLuTK.COX2 transductional retargeting to CRC xenograft metastases will be optimized with sCAR-f-alphaCEA, a recombinant molecule containing soluble CAR, the phage T4 fibritin trimerization domain and a single-chain anti-CEA antibody. Combined sCAR-f-alphaCEA transductional liver untargeting, transductional tumor retargeting and COX-2 restricted expression should optimize fLucTK expression in CRC hepatic metastases. HSV1-TK/ganciclovir therapy should then effectively eradicate CRC metastases, with minimal side effects. We will employ bioluminescent imaging to monitor restricted fLucTK expression, bioluminescent imaging from xenografts marked with Renilla luciferase and 124I-antiCEA minibody/microPET imaging to monitor tumor burden during HSV1- TK/GCV therapy, and FDG and FLT/microPET to monitor early therapeutic responses. Our goal is to optimize this therapeutic protocol for CRC hepatic metastases, in apreclinical model of human disease.

The future of prostate cancer research lies in the continued creation and development of translational projects and innovative investigators at major research institutions, as well as an institutional commitment to support outstanding, dedicated scientists in the field. In order for new physician-scientists to grow and flourish, funding support and peer mentorship is imperative to this process. The SPORE Career Development Program will provide this research support and mentoring resource, allowing junior faculty to transition to independent investigator status with adequate peer-reviewed funding. Career Development Program funds will be designated to prepare investigators for careers in translational prostate cancer research. In addition, in well-justified selected cases, mid-level and senior investigators who are already established in another area of research will be eligible for support to re-direct their research focus to prostate cancer.

Adenovirus vectors are among the most popular vehicles for cancer gene therapy protocols. However, adenovirus infect cells via the Coxsackie and Adenovirus Receptor, which is often more extensively expressed on normal cells then on tumors. We utilized bioluminescent imaging following systemic Ad.CMVfLuc administration to monitor adenovirus transductional "untargeting" and "retargeting" by sCAREGF. sCAR-EGF is a bi-specific retargeting molecule containing the soluble portion of CAR linked to epidermal growth factor. Noninvasive optical imaging demonstrates that systemically injected [Ad.CMVfLuc] [sCAR-EGF] complexes have reduced ability to infect liver, but can infect EGF receptor-positive xenografts. COX-2 is not expressed in most normal tissues, but is overexpressed in many tumors. We used noninvasive optical imaging to examine fLuc expression following intratumoral injection of Ad.COX2fLuc (an adenovirus expressing luciferase from the COX-2 promoter). No expression occurs in liver. In contrast, expression occurs in COX-2 positive tumors. Colorectal cancer (CRC) is a leading cause of cancer death. CRC frequently metastasizes to liver. CRC liver metastases often over-express both carcinoembryonic antigen (CEA) and COX-2. We will combine our experience - both with transductional "untargeting" and "retargeting", and with restricted COX-2 gene expression - to develop effective therapies for CRC hepatic metastases. We will establish xenograft CRC models of liver metastasis. We will create Ad.COX2.fLucTK.COX2, an adenovirus that expresses a fusion protein containing both firefly luciferase and HSV1-thymidine kinase (fLucTK). CRC tumor-restricted fLucTK expression will result from regulation by both the COX-2 promoter and the COX-2 mRNA 3' untranslated region. Transductional liver untargeting and Ad.COX2.fLuTK.COX2 transductional retargeting to CRC xenograft metastases will be optimized with sCAR-f-alphaCEA, a recombinant molecule containing soluble CAR, the phage T4 fibritin trimerization domain and a single-chain anti-CEA antibody. Combined sCAR-f-alphaCEA transductional liver untargeting, transductional tumor retargeting and COX-2 restricted expression should optimize fLucTK expression in CRC hepatic metastases. HSV1-TK/ganciclovir therapy should then effectively eradicate CRC metastases, with minimal side effects. We will employ bioluminescent imaging to monitor restricted fLucTK expression, bioluminescent imaging from xenografts marked with Renilla luciferase and 124I-antiCEA minibody/microPET imaging to monitor tumor burden during HSV1- TK/GCV therapy, and FDG and FLT/microPET to monitor early therapeutic responses. Our goal is to optimize this therapeutic protocol for CRC hepatic metastases, in apreclinical model of human disease.

DESCRIPTION (provided by applicant): Clinical evidence, epidemiological results and experimental animal studies overwhelmingly suggest that COX-2 over-expression plays a modulatory and even causal role in development of many epithelial cancers. Recent studies demonstrate that stromal fibroblasts and blood vessel endothelial cells modulate epithelial tumor formation. There also exist substantial correlative data suggesting that COX-2 expression in stromal fibroblasts and blood vessel endothelial cells - and not in the initiated epithelial tumor precursor cells - may mediate pre-neoplastic emergence and subsequent progression of epithelial cancers. However, existing COX-2 pharmacologic experiments and global Cox2 knockout mice do not permit either physiological or genetic analyses to determine the specific cell populations - fibroblasts, epithelial or blood vessel endothelial cells - in which COX-2 over expression plays a critical role(s) in tumor development. We have developed (i) COX2 COE transgenic mice, in which we can conditionally over-express COX-2 in targeted cells and tissues and (ii) Cox2flox mice, in which we can conditionally delete the Cox2 gene in targeted cells and tissues. By crossing COX2 COE mice and Cox2flox mice to transgenic mice in which a tamoxifen-regulated CreERT recombinase is expressed in either epithelial cells, stromal fibroblasts or blood vessel endothelial cells, we will be able to determine (1) whether COX-2 over production in epithelial cells, fibroblasts and/or blood vessel endothelial cells modulates cancer development and (2) whether COX-2 expression is required in epithelial cells, fibroblasts or blood vessel endothelial cells for cancer development. Skin cancer is among the best-studied epithelial tumor induction models. We chose skin cancer to study the role(s) of COX-2 in epithelial cells, stromal fibroblasts and blood vessel endothelial cells during development of epithelial cancer because of the extensive literature on this model, the ease with which tumors can be monitored non-invasively, and the ease with which CreERT can be activated locally. The "broad, long-term objectives and specific aims" of this application are to determine (1) whether COX-2 plays a modulatory and/or a required role(s) in development of epithelial cancer and (2) in which cell(s) - initiated epithelial tumor precursor cell and/or stromal fibroblast and/or blood vessel endothelial cell - COX-2 modulates either pre-malignant epithelial tumor development or progression of benign tumors to carcinomas. The relevance of this research to public health. COX-2 inhibitors are still investigated as therapeutic and preventive agents for epithelial cancers, despite cardiovascular side effects. "Down-stream" COX-2 pathway effectors (prostanoid synthases and receptors) have become targets for similar research. If we can pinpoint the cell type(s) in which COX-2 expression and the prostanoid effectors regulate cancer development, we may be able to target those steps with lower concentrations and less prolonged application of pharmacologic agents. We anticipate we will be able to define the critical cells and times for COX-2 cancer enhancement.

The Small Animal Imaging Shared Resource (SAISR) was established as a JCCC Shared Resource in 2002, with a prototype microPET scanner, a microCT, a digital whole body autoradiograpy (DWBA) system and an optical imaging system. Twelve JCCC faculty formed the user base in 2002. During the past five years all initial SAISR instrumentation has been replaced; we currently have a dedicated imaging suite that houses a new microCT, two microPET instruments, a new cryostat and DWBA system, three bioluminescence/fluorescence optical imaging instruments and a Maestro spectral filtering fluorescence system. All instrumentation was obtained with institutional support. The SAISR has a dedicated vivarium for bngitudinal imaging studies. Our user base has grown to 44 faculty members who performed over 14,000 imaging experiments last year. JCCC members from seven Program Areas performed 86% of these studies. JCCC CCSG support for the SAISR is 14% of the SAISR operating budget. During 2008 the SAISR will move into a new 1500-sq. ft. imaging laboratory in the California NanoSystems Institute building. This facility will include a dedicated vivarium, procedure rooms, microPETs, microCT, bioluminescence, fluorescence and DWBA capabilities and adjacent space for a cyclotron dedicated to small animal imaging research and for radiochemistry facilities. This new physical facility is a tangible demonstration of institutional support for the SAISR in particular and the JCCC in general. The SAISR is full service, providing didactic classroom instruction and training in imaging instrumentation and animal handling protocols, individual instruction, online instruction, standard operating procedures for imaging procedures, on-line remote scheduling for instrumentation, data storage and archiving, data retrieval and data processing, on-site data retrieval for staff consultation and assistance in data processing, all probes and contrast reagents for microPET, microCT and optical imaging experiments, anesthetics, miscellaneous supplies (syringes, heating platforms, surgical materials, isolators for immunocompromised mice, multimodality imaging chambers designed by SAISR staff) and software for data analysis. Assistance is also provided for animal protocol preparation, radiation safety approval, data analysis and presentation and co-registration of multimodality images. All instruments are maintained and calibrated by SAISR staff. Critical staff, who have a long association with the JCCC, the SAISR and the investigators, provide both technical assistance and experimental guidance and expertise. In addition to assisting investigators with specific experimental protocols, the SAISR provides support to the investigators in the development and translation of new instrumentation, probes and contrast agents, and assists with biodistribution, dosimetry and toxicology studies. (Please also see Section 6.2.3 on Shared Resources in the History, Description, Essential Characteristics). 29 Cancer Center members representing eight Cancer Center Program Areas utilized the services of the Small Animal Imaging Shared Resource during the reporting period. This is a continuing shared resource.

Colorectal cancer (CRC) kills 50,000 Americans every year. Half have hepatic metastases at diagnosis;only a small percentage can be cured. Our overriding goal is to develop new tools to deliver therapeutic genes to hepatic CRC metastases and new tools to monitor non-invasively therapeutic gene delivery and efficacy. CRC tumors often overexpress both carcinoembryonic antigen (CEA) and cyclooxygenase 2 (COX-2). Hepatic metastases generally express even greater levels of CEA and COX-2 than their primary tumors. The aims of our currently funded Research Component 4 project are to: (1) Establish 00X2"" CEA* CRC hepatic xenografts that can be monitored by non-invasive imaging, as models of hepatic CRC metastasis. (2) Create AdCox2fLucTK, an adenovirus from which a firefly luciferase/HSVI-TK fusion reporter-therapeutic protein is regulated by the Cox2 promoter. (3) Characterize sCAR-aCEAs, adenovirus liver-untargeting/CEA* tumor-retargeting agents that fuse the Coxackie and Adenovirus Receptor (CAR) extracellular domain to a single-chain anti-CEA antibody. (4) Examine the ability of (i) sCAR-aCEA reagents to retarget Ad gene delivery vectors to CEA* CRC hepatic xenografts and the ability of (ii) Cox2 regulatory elements in Ad vectors to restrict transgene expression to COX-2* CRC xenografts, and (5) Optimize ganciclovir-dependent killing of hepatic C0X2* CEA* CRC metastases by intravenously administered, Cox2 transcriptionally restricted, transductionaily re-targeted [Ad.Cox2fLucTK][sCAR-aCEA]. We accomplished all these goals. This project received the lowest (best) priority of the current ICMIC Research Component projects. However, several criticisms from the Summary Statement form the basis of this renewal application. What are: (1) The effects of innate immunity on transductional retargeting and transcriptional restriction? (2) The effects of preexisting immunity on Ad transductional retargeting and transcriptional restriction? (3) The effects of shed, circulating soluble CEA on Ad retargeting? (4) The final criticism was a lack of a plan for clinical translation. Our Specific Aims are: (1) To establish C0X2* CEA* hepatic CRC models in immunocompetent B57BI6 and CEA-transgenic mice, and to use this model to (2) minimize the innate immune response to transcriptionally restricted/transductionally retargeted Ad imaging and therapeutic vectors, (3) optimize Ad vector evasion of neutralizing anti-ad antibody, and (4) optimize inhibition of sCAR-aCEA retargeting of Ad vectors to CEA* hepatic CRC metastases by shed CEA. (5) Our final aim is to design, develop reagents for, obtain approval for and initiate a clinical trial of Ad.Cox2HSV1sr39tk/FHBG PET imaging of CRC metastases in patients.

Overview of the organizational structure of the UCLA ICMIC (Figure 1). Dr. Herschman is Principal Investigator of the UCLA ICMIC, Dr. Phelps will serve functionally as Co-Principal investigator, although this is no longer a recognized NIH title. Drs. Herschman and Phelps are supported by a budgeted Administrative Component that include secretarial, financial and grants preparation assistance. The ICMIC program has four Research Component projects, headed by Drs Ribas, Radu, Hong Wu and Herschman. There are three Specialized Resources. The Cyclotron and Radiochemistry Specialized Resource, directed by Dr. Satyamuthy, provides positron-emitting radiotracers for ICMIC studies. The Molecular Imaging Specialized Resource, directed by Dr. Stout, provides access to molecular imaging instrumentation as well as expertise, instruction and technical assistance in their utilization. The Quantitative Image Analysis Specialized Resource, directed by Dr Henry Huang, provides expert assistance in image interpretation and quantization, data storage and retrieval, and modeling of microPET data. The Specialized Resources provide imaging reagents, instrumentation, technical assistance, instruction and intellectual insight in support of the research efforts of the Research Component Projects, the Developmental Fund Projects and the Career Development recipients. The Developmental Fund program supports a steady state of four Developmental Projects, usually of two years duration. We try to stagger the projects so that each year there will be a competition for two new Developmental Fund Projects. However, second year applications are in competition with new applications, and on occasion we will terminate a Developmental Project at the end of the flrst year and initiate three new Developmental Projects in one year. Our Career Development Component provides individual stipends to candidates who want to integrate molecular imaging into their research programs- Career Development funds are recompeted annually. Drs. Herschman and Phelps are advised quarteriy by the Internal Advisory Board (Section B.4), which is made up of past and present ICMIC project directors, and annually by members of the External Advisory Board.

CAREER DEVELOPMENT COMPONENT OF THE UCLA-ICMIC. F.I. The UCLA-ICMIC Career Development Component has been, and will continue to be, a flexible program. Our goal is to train outstanding candidates who have an interest in the interdisciplinary interface of basic cancer research and/or clinical-translational studies with molecular imaging applications. At the end of their ICMIC support, we hope their training will contribute to careers that increase cancer discovery research or improve diagnosis, prediction of response to therapy and/or monitoring of therapy. We are as flexible as possible with regard to the spectrum of trainees that we will recruit and support. However, we make - as a condition of support from Career Development resources - a provision that recipients participate in didactic instruction either in Cancer Biology or in principles of Biological Imaging (or both!) if the review committee is of the opinion that the candidate has a deficiency that will not be corrected by laboratory participation alone. Our Internal Review Board, made up of our Research Component P.l.s (Drs. Hong Wu, Caius Radu, Antoni Ribas), Drs. Herschman and Phelps, and Drs. Owen Witte and Jonathan Braun review applications and select trainees. We will also require, as a condition of support, that individual trainees participate in portions (tailored to the individual) of a curriculum that includes specific courses, individual laboratory research group meetings, participation in Crump Institute for Molecular Imaging, Jonsson Comprehensive Cancer Center and Institute of Molecular Medicine internal seminars, and UCLA-ICMIC weekly research meetings. We will also request of trainees and their mentors that trainees receive some practical experience in all the imaging modalities available in our Specialized Resources. Our goal, in addition to a successful research project, will be that - at the conclusion of their support period - the trainees will be familiar with the fundamental principles of biological imaging and cancer biology, and expert in one or more areas of imaging and cancer research. While we will not expect trainees to have mastered radiosynthetic chemistry, mathematical reconstructions of tomographic images and all the other technologies required for their research in this interdisciplinary program, we will expect a working knowledge of all these areas and expertise in their areas of concentration.

DESCRIPTION (provided by applicant): Novel molecular imaging approaches to monitor gene and cell-based therapies Gene and cell-based therapies have ushered in a new era of opportunities in regenerative medicine and oncology. However, a critical roadblock in the effective development and evaluation of cellular therapeutics is the inability to follow the fate and function of the therapeutic genes and cells in treated patients. We propose to develop technologies for specific identification and tracking of therapeutic genes and cells in vivo using positron emission tomography (PET), a quantitative, non-invasive molecular imaging approach applicable to both preclinical and clinical settings. This application addresses a key limitation of current reporter gene strategies, in which therapeutic vectors and cells are genetically modified to produce a signal detectable by PET. Instead of commonly used, highly immunogenic viral proteins, we will generate novel PET reporter genes based on fully human proteins, to overcome this challenge to clinical implementation. This project relies on many years of grants and basic research results that are now ready to advance to the commercial domain. We propose a three year effort to turn recent advances into practical outcomes delivered as end-user-ready PET Reporter Gene (PRG) delivery kits and PET Reporter Probes (PRP) that will enable whole body pharmacokinetic and therapeutic outcomes information. This application will also deliver preliminary information from a first-in-human Phase 0 small trial of new PET reporter probe biodistribution and dosimetry. Our proposal leverages an established partnership between UCLA (the laboratories of the Ahmanson Translational Imaging Division and the laboratory of Harvey Herschman) and CellSight Technologies (CST, a biotech company based in San Francisco, CA). The UCLA-CST partnership builds on past extensive interactions at UCLA between investigators and consultants, as described in this application. We will carry out four Specific Aims. Aim 1 consists of in vitro, cell culture and animal studies to evaluate and optimize new PET Reporter Gene-PET Reporter Probe (PRG-PRP) systems. Our new current PRG being developed is a point mutant (N44D) of the human thymidine kinase 2 (tk2) gene. L-[18F]FMAU and L- [18F]FEAU, two hTK2-N44D substrates, are our new PRPs. In Aim 1 we will also determine whether the TK2- based PRG can elicit an immune response in humans and we propose a strategy to eliminate this possibility. In Aim 2 we propose a stringent preclinical evaluation of the new PRG-PRP systems, using animal models of gene and cell-based therapies against two types of cancer: hepatic metastases of colorectal cancer and melanoma. Aim 3 proposes a strategy to develop, validate, and commercialize kits for PRG delivery into murine and human therapeutic cells and a plan to disseminate this new capability to wider communities of end- users. In Aim 4 we will complete an eIND submission to enable first-in-human studies of the biodistribution and dosimetry of the new PET reporter probes L-FMAU and L-FEAU. These first-in-human studies will set the stage for a follow-up study in which UCLA and CST will submit a full IND application to the FDA to initiate clinical testing of the new PRG-PRP systems in cancer patients. The set of new PET imaging technologies co-developed by UCLA and CST investigators may find immediate clinical applications in experimental gene and cell-based therapies in cancer and may be broadly applicable to therapies for diseases with significant public health impact, including transplantation of hematopoietic stem cells in congenital and acquired disorders such as AIDS, islet cells in type 1 diabetes, ES-derived neural stem cells in Parkinson's disease, and stem cells in myocardial dysfunction.