Raymond N. DuBois - US grants

Overall, our long-term goal is to obtain a better understanding of the regulation of renal epoxygenase enzymes and determine the role they play in renal function. The presence of an active cytochrome P450 monooxygenase in renal tissue is well documented. Even though the kidney contains the second highest body concentration of cytochrome P450 enzymes, relatively little is known about the catalytic and molecular properties of the renal hemoproteins. In earlier studies it was shown that, in addition to catalyzing lauric acid omega/omega-1 oxidation, kidney cytochrome P450 was active in the metabolism of several drugs and carcinogens. The inducibility and the segmental distribution of specific cytochrome P450 isoforms along the nephron is well established. The initial demonstration of a role for cytochrome P450 in the omega/omega-1 oxidation of prostanoids and leukotrienes, known urinary metabolites of these functionally important mediators, suggested a physiological role for this enzyme system. More recent studies have documented a function for renal cytochrome P450 as an active catalyst for the metabolism of arachidonic acid to biologically active products. The specific aim of this project is to isolate and characterize genes encoding cytochrome P450 2C (epoxygenase) family members from rat kidney and determine the molecular basis for the effect of dietary salt loading on this enzyme system. Preliminary evidence indicates that this family of cytochrome P450 isoforms is involved in the metabolism of endogenous substrates (arachidonic acid) to bio-active compounds which affect renal vascular tone, free water and ion transport. The enzymatic activity of one of these isoforms has been shown to be dramatically induced in the kidney after placing rodents on a high salt diet. We propose to clone and sequence the control and salt-regulated renal epoxygenase genes and then study the molecular basis for the increase in the level of epoxygenase activity in animals given a high salt diet. In addition, other preliminary evidence outlined in project 2 demonstrates that in the Dahl+ salt sensitive strain of rats there is a lack of induction of epoxygenase activity following salt loading. We also propose to clone the homologue of the salt regulated epoxygenase (SREP) gene from the Dahl+ rat kidney and determine the molecular basis for the absence of salt regulation of this enzyme in the Dahl+ (salt sensitive) rats.

The gastrointestinal tract provides an excellent model system in which to study the regulation of growth and differentiation. The continuous process of intestinal epithelial proliferation and differentiation is tightly regulated and organized into specific compartments. The molecular mechanisms which underlie intestinal epithelial growth regulation and differentiation are poorly understood. Our broad long-term goal is to obtain a better understanding of the molecular basis of intestinal epithelial growth regulation and transformation. Recently our laboratory cloned an intestinal mitogen inducible cyclooxygenase gene which is upregulated by transforming growth factor alpha (TGFalpha) and tumor promoters. Treatment of rat intestinal epithelial cells with cyclooxygenase inhibitors blocks TGFalpha stimulated mitogenesis. The recombinant RS-2 protein has cyclooxygenase activity which is completely inhibited by sulindac sulfide, a potent non steroidal anti-inflammatory drug (NSAID). Treatment of the recombinant enzyme with another commonly used NSAID (aspirin) converts it from a cyclooxygenase to a lipoxygenase which produces almost exclusively 15(R)-HETE, a novel eicosanoid product. Several animal and epidemiologic studies demonstrate that NSAID use is linked to a 50-60% reduction in colon cancer. The mitogen inducible cyclooxygenase mRNA is increased in some human colon cancers. What is the role of the inducible cyclooxygenase gene and the eicosanoid products it produces in intestinal epithelial growth regulation and transformation? What are the molecular mechanisms regulating its expression? We propose to sequence the full length mitogen inducible cyclooxygenase-related gene (RS-2) from growth factor stimulated intestinal epithelial cells and prepare large quantities of the RS-2 recombinant protein for further characterization. We are particularly interested in determining if selected NSAIDs preferentially inhibit RS-2. We will isolate and characterize the 5' flanking sequences of the RS-2 gene and determine the TGFalpha and eicosanoid cis regulatory sequences in the Zif268 and RS-2 promoters. This will give us a better understanding for the molecular basis for the regulation of the induction of these genes in intestinal epithelial cells. We will determine the role of RS-2 in intestinal epithelial growth regulation and transformation utilizing antisense and transgenic approaches. We will design antisense oligonucleotides to specifically inhibit the expression of RS-2 and/or Zif268 and then determine what effect this has on TGFalpha-induced eicosanoid production and mitogenesis. This will help to precisely determine the importance of these genes in intestinal epithelial growth regulation. We will link the RS-2 coding region downstream of the villin and metallothionine promoters and prepare transgenic mice which will overexpress this gene in various regions of the intestine and then determine what effect this has on growth regulation. We will add an initiator to transgenic mice and determine if tumor formation is accelerated in mice overexpressing the RS-2 gene relative to control mice. These studies should provide insights about the role of the mitogen inducible cyclooxygenase gene and the eicosanoids it produces in intestinal epithelial growth regulation, differentiation and transformation.

DESCRIPTION: (Applicant's Description) Colorectal cancer is the second leading cause of cancer deaths in the U.S. and therefore, a major public health issue. Prevention or control of colorectal cancer could result in a significant saving of lives and health care dollars. Several studies have reported a 40-50% reduction in the risk of developing colorectal cancer in persons who take nonsteroidal anti-inflammatory drugs (NSAIDs), like aspirin, on a regular basis. One possible mechanism for this NSAID-effect to reduce cancer risk is via the inhibition of cyclooxygenase enzymes. Two isoforms of this enzyme have been characterized, which will be referred to as cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) in this proposal. COX-2 expression is markedly increased in 40-50% of adenomas and in 85-90% of human colorectal adenocarcinomas, while COX-1 levels remain unchanged. Most of the NSAIDs examined to date in clinical studies inhibit both the COX-1 and COX-2 enzymes. Controversy exists over the role of cyclooxygenase enzymes in the prevention of colorectal cancer. Some groups do not feel that the cyclooxygenase pathway plays an important role because cell culture experiments have shown that one NSAID, lacking COX inhibitory activity, can induce apoptosis in cells which lack expression of the COX-2 gene. These effects are only seen at drug concentrations above the range of 400-750 micromolar. Nonetheless, this work points out the importance of maintaining an objective approach and designing experiments to evaluate the mechanisms involved in the chemoprotective activity of NSAIDS. To address this controversy, we plan to carry out the studies described in Project 1 to determine the relative importance of the cyclooxygenase pathway in the regulation of programmed cell death by NSAIDs. We plan to employ animal models to test the hypothesis that COX-2 and/or COX-1 may be relevant targets of NSAID drugs in the prevention of colorectal cancer. Our specific aims are to: 1) Utilize xenograft models in athymic nude mice to evaluate the mechanism by which selective and non-selective NSAIDs cause regression of tumors positive and negative for COX-2 expression, 2) Utilize mouse lines lacking either COX-1 or COX-2 expression to determine the relative importance of the presence of either enzyme in NSAID-induced apoptosis in the intestine, 3) Crossbreeding of COX-1 and COX-2 null mice with Min and APC mutant mice to determine the importance of COX enzymes in intestinal tumorigenesis and whether treatment with NSAIDs could further reduce tumor number in COX deficient mice. By utilizing these approaches our overall goal is to better understand the mechanisms involved in the chemoprevention of colorectal cancer and whether highly selective NSAIDs could be effective agents in the chemoprevention of intestinal cancer.

OVERALL DESCRIPTION (Applicant's Description) The overall goal of this Program Project Grant is to determine the molecular mechanisms involved in the chemoprevention of colorectal cancer by non- steroidal anti-inflammatory drugs (NSAIDs). In this regard, we will specifically test the hypothesis that the cyclooxygenase (COX) pathway and/or its eicosanoid products play a role in colorectal carcinogenesis. Colorectal cancer is the second leading cause of cancer related deaths in the United States. This year alone, approximately 55,000 Americans will die from this disease. Recent clinical research has revealed a 40-50% reduction in mortality from colorectal cancer in persons who take aspirin and other NSAIDs on a regular basis. Cyclooxygenase enzymes are known targets for NSAIDS. Two isoforms of cyclooxygenase have been characterized and they are referred to as Cyclooxygenase-1 (COX-1) and Cyclooxygenase-2 (COX-2) in this proposal. Previous work conducted by investigators involved in this program grant has demonstrated that there is a 2-50 fold increase in COX-2 expression in 85% of human colorectal adenocarcinomas and in 45-50% of adenomas. The goals of this program are to test the hypothesis that COX or its eicosanoid products play a role in colorectal carcinogenesis and to determine the molecular mechanisms by which NSAIDs prevent colorectal cancer. Here we provide an overview of the projects proposed to test this hypothesis and highlight how planned interactions among the investigators will aid significantly in the success of this program project. In addition, the role of COX-2 and prostaglandins in the pathogenesis of colorectal cancer is discussed, so as to provide a rationale for undertaking the projects that are proposed. There are four projects and three cores included in this revised Program Grant application.

DESCRIPTION (provided by applicant): This application is being submitted to obtain funding for the Vanderbilt Digestive Disease Research Center (VDDRC). Our Center?s major theme is the study of the molecular and cellular mechanisms responsible for digestive diseases. The VDDRC is multidisciplinary, including faculty in 12 different academic departments with 45 investigators (30 full members and 15 associate members). The aims of the VDDRC are aligned with the goals of Vanderbilt University: 1) to promote digestive diseases-related research in an integrative, collaborative and multidisciplinary manner; 2) to enhance the basic research capabilities of VDDRC Members; 3) to attract investigators not involved in digestive diseases-related research to pursue these lines of investigation; 4) to develop and implement programs for training and establishment of young investigators in digestive disease-related research; and 5) to facilitate the transfer of basic research findings to the clinical area. Investigative interests of the members fall into four broad areas of study: 1) growth regulation: proliferation, differentiation, and apoptosis, 2) maintenance of epithelial integrity, 3) enteric neuroscience, and 4) GI physiology and metabolism. The VDDRC contains four core research laboratories to support the members: 1) the Microarray Core, 2) the Cellular and Animal Modeling Core, 3) the Cell Imaging Core, and 4) the Bioanalytical (Mass Spectrometry) Core. These core laboratories were integrated into our Center to provide investigators working on digestive disease-related research with the latest advancements in technology and aid in experimental design. The VDDRC supports a Pilot/ Feasibility Program and a Young Investigator Award Program to foster participation of beginning and seasoned investigators in research related to digestive diseases through its Administrative Core. This Core also contains Biostatistical and Enrichment Programs and oversees the financial management and operation of the VDDRC.

[unreadable] DESCRIPTION (provided by applicant): Carcinoma of the colon and rectum is the second-leading cause of cancer related deaths in the United States and thus represents a major public health concern. The disease develops due to a loss of the normal regulatory pathways that govern the transition from undifferentiated stem cell to mature colon epithelial cell. The nuclear hormone receptor (NHR) super family of transcription factors are important regulators of many biological pathways including cellular differentiation. One family of NHRs, the peroxisome proliferator-activated receptors (PPARs), is activated by fatty acids and certain fatty acid metabolites and plays important role in metabolic homeostasis. For example, the PPAR subtype is a central regulator of adipocyte differentiation and synthetic agonists of the receptor enhance insulin sensitivity and are being widely used in the treatment of type ll diabetes mellitus. We and others have found PPAR to be strongly expressed in post-mitotic, differentiated colon epithelial cells and have demonstrated that activation of PPAR in a broad spectrum of colorectal cancer cell lines induces growth inhibition associated with a delay in the 01 phase of the cell cycle. Activation of the receptor also leads to an increase in several markers of intestinal epithelial cell differentiation. Consistent with an anti-oncogenic, pro-differentiation role for PPAR in the colon, loss of function mutations in the receptor have been identified in a subset of colorectal tumors and cell lines. Collectively, these findings raise the possibility that PPAR agonists may have clinical value as a form of differentiation therapy for colorectal cancer. However, despite these advances our understanding of PPAR signaling in the colon remains superficial. The major focus of this proposal is to define the mechanistic basis for the biological phenotype induced by PPAR activation in colorectal cancer cells. Specifically, we hypothesize that binding of ligand to apo-PPAR causes conformational changes in the receptor leading to the recruitment of a specific set of co-regulatory molecules that then leads to the regulation of target genes that are important modifiers of colon epithelial cell growth and differentiation. Our experimental approach will focus on 1) identifying the structural domains unique to the PPAR subtype that allows the ligand-bound receptor to induce colorectal cancer cell differentiation; 2) identify the co-regulators critical for PPAR-mediated differentiation specifically in the colon; and 3) define the role of the PPAR target gene transforming growth factor-stimulated clone-22 (TSC-22) in intestinal epithelial cell function. Results from these experiments have the potential not only to lead to the design of PPAR activators with enhanced anti-tumor efficacy, but also help clarify the molecular basis of intestinal epithelial cell differentiation.

DESCRIPTION (provided by applicant): This is the second competing continuation application for the Vanderbilt-Ingram Cancer Center (VICC) CCSG. The VICC is a matrix center within Vanderbilt University Medical Center (VUMC), and the VICC integrates the cancer-related expertise and resources of the School of Medicine, School of Nursing, School of Arts and Sciences, School of Engineering, and Peabody School of Education as well as the fully integrated Veterans Administration Medical Center (VAMC). All facilities are located on the same campus, a situation that promotes interactions, sharing of resources, and collaborations. Established in 1993, the VICC functions as an organizational unit with a supradepartmental status. The VICC's specific authorities and responsibilities are: 1) to conduct coordinate and integrate the cancer and cancer-related activities of Vanderbilt University; 2) to conduct, support and enhance cancer research and to integrate cancer-related activities throughout the University; 3) to integrate, develop and conduct cancer education programs; and 4) to coordinate and integrate the care of cancer patients at VUMC and VAMC. The research objectives are accomplished through seven Research Programs that are essentially the same as in the previous application with minor name changes. The Programs are: Signal Transduction and Cell Proliferation, Cancer Proteomics and Genomics, Host-Tumor Interactions, Gastrointestinal Cancer, Breast Cancer, Cancer Prevention and Population-Based Research, and Experimental Therapeutics. Sixteen Shared Resources are proposed representing eight previously supported and eight new. There has been remarkable progress in the development of the VICC over the past project period. The Center has been renamed the Vanderbilt-Ingram Cancer Center based on a substantial commitment of philanthropic funds from the Ingram family that initiated a capital campaign. Funds from the capital campaign have been leveraged with Institutional funds to recruit 80 new faculty enhancing all of the VICC Research Programs, establish 20 endowed professorships for recruitment and retention, expand space controlled and utilized by the VICC, and establish cutting-edge genomic, proteomic and informatics shared resources. A large population-based research initiative was established through recruitment of 12 cancer epidemiologists who now head three newly funded major cohorts based at the VICC, the Southern Community Cohort, the Women's Shanghai Cohort, and the Men's Shanghai Cohort. Three NCI SPORE grants have been funded, and the NCI funding base for the VICC has increased 2.9 fold since the last renewal.

The COX-2 enzyme, induced during various modes of cell activation, catalyzes the rate-limiting steps in the conversion of cellular lipids into prostaglandins (PG). Recent data strongly implicate the critical role of COX-2 pathway in cancer development and progression. Complex cellular mechanisms control the precise regulation of expression of the COX-2 gene; for example, regulation at transcriptional and post-transcriptional levels is important. Once induced, the COX-2 enzyme couples to specific downstream pathways, such as the cell surface PG receptor system or the nuclear peroxisomal proliferators activator receptors (PPAR). In the context of tumor biology, the COX-2 enzyme metabolizes multiple substrates, for example, free arachidonate and endocannabinoids. This proposal will examine mechanisms of COX-2 regulation and function in the context of tumor biology. The first aim of the proposal is based on the hypothesis that the COX-2 enzyme is aberrantly induced in tumors due in part to the function of the RNA binding protein HuR. We have recently shown that the RNA binding protein HuR regulates COX-2 gene expression at the level of mRNA transport and stability. We will further explore the mechanisms of how HuR achieves the stabilization of COX-2 mRNA. We will also examine if HuR expression or activity is critical in exaggerated COX-2 that occurs in cancer. The transgenic mouse model of COX-2 induced mammary tumor formation will be used to obtain in vivo correlates of HuR involvement in COX-2 mRNA expression. The second aim of the proposal seeks to obtain novel mechanistic insights into COX-2 function. It is generally accepted that the COX-2 enzyme couples to the cell surface PG receptor and the nuclear PPAR receptors. Precisely how this is achieved and how cellular controls are exerted to differentially regulate COX-2 coupling to different effector pathways is poorly understood. We hypothesize that differential substrate utilization (arachidonate versus endocannabinoids) is a mechanism that determines if COX-2 couples to the cell surface versus nuclear receptor pathways. Furthermore, differential expression and subcellular localization of effector systems may determine coupling to the COX-2 pathway. These issues will be investigated in cell culture systems of endothelial cells, mammary epithelial cells and the transgenic model of mammary tumorigenesis. Overall, the proposed lines of investigation will likely procure additional mechanistic insights relevant to the role of COX-2 in tumor biology. Synergy of PPG cores and projects is anticipated to greatly enhance this particular project. In addition, this project will contribute to the PPG by providing unique insights into the role of COX-2 in mammary tumorigenesis. Such efforts may ultimately lead to novel mechanism-based chemopreventive approaches in human cancer.

Colorectal cancer (CRC) is a significant health concern in most industrialized countries. A significant effort has been made in the identification of drug targets for both the prevention and treatment of colorectal cancer over the past decade. Epidemiological, experimental, and clinical studies have demonstrated that there is a significant reduction in risk for colorectal cancer in persons who take nonsteroidal anti-inflammatory drugs (NSAIDs). A large body of genetic and biochemical evidence has demonstrated that the cyclooxygenase-2 (COX-2) enzyme is a promising NSAID target for prevention of colorectal cancer. In COX-2-dependent pathways, NSAIDs suppress colon carcinogenesis through the inhibition of prostaglandin formation. Studies by our group have demonstrated that 1) a synthetic PPARdelta agonist increases the number and size of polyps in Min mice; 2) COX-2 derived PGE2 induces PPARdelta transactivation; 3) PGE2 induces colon cancer cell migration/invasion through a EGFR-PI3k-Akt pathway; 4) NSAIDs downregulate a novel gene NRG-1 (NSAID regulated gene-1), which is highly expressed in human colorectal tumor tissue and is up-regulated by Ras. However, the precise mechanisms for the COX-2-dependent or -independent effects of NSAIDs on colorectal carcinogenesis are not clear. The experiments we propose here will explore these issues further and attempt to understand the mechanisms by which NSAIDs affect colorectal biology. Our experimental approaches will focus on the following three specific aims: 1) Determine the biologic function of PPARdelta in the intestinal polyp formation; 2) Characterize downstream pathways of COX-2 derived PGE2 in colorectal cancer biology; and 3) Characterize the biologic function of the NSAID-regulated gene-1 (NRG-1) in colorectal cancer formation.

DESCRIPTION (provided by applicant): The overall goal of this Program Project Grant is to determine the molecular mechanisms involved in the chemoprevention of cancer by non-steroidal anti-inflammatory drugs (NSAIDs). We will specifically test the hypothesis that the cyclooxygenase (COX) pathway and/or its eicosanoid products play a role in certain aspects of breast, cervical, ovarian and colorectal carcinogenesis. These solid malignancies lead to a large number of deaths each year. Better detection and prevention strategies are needed. NSAIDs have effects on model systems for these diseases. Cyclooxygenase enzymes are known targets for NSAIDs. Two isoforms of cyclooxygenase have been characterized and they are referred to as Cyclooxygenase-1 (COX-1) and Cyclooxygenase-2 (COX-2) in this proposal. The goals of this program are to test the hypothesis that COX enzymes or their eicosanoid products play a role in carcinogenesis and to determine the molecular mechanisms by which NSAIDs prevent colorectal cancer and prostaglandin production increases risk for neoplasia. Here we provide an overview of the projects proposed to test this hypothesis and highlight how planned interactions among the investigators will aid significantly in the success of this program project. There are four projects and three cores included in this competing renewal Program Project Grant application. Research Projects 1. Role of COX downstream signaling pathways in cancer 2. COX-2: A target for the prevention of cervical cancer 3. COX-2 regulation and function in tumor biology 4. COX-1: A target for ovarian cancer prevention and treatment Cores A. Administrative Core B. Eicosanoid Analysis Core C. Animal and Molecular Biology Core

[unreadable] DESCRIPTION (provided by applicant): There are several molecular signaling pathways involved in the regulation of intestinal homeostasis and epithelial differentiation that are important in a number of digestive diseases. Colorectal cancer is one such disease and is a major health concern in this country. One group of compounds found to decrease the risk of colorectal cancer is non-steroidal anti-inflammatory drugs (NSAIDs). However, prolonged use of some of these agents is associated with unacceptable side effects (cardiovascular and gastrointestinal). Thus, it is crucial to develop more effective preventive measures with minimal toxicity and maximum benefit. Peroxisome proliferator-activated receptors (PPARs) are potential targets for chemopreventive agents. There are three PPAR isoforms, PPARa, PPARd, and PPAR?, which belong to the nuclear hormone receptor superfamily. Preliminary data indicates that a PPARd agonist promotes intestinal adenoma growth, increases tumor cell survival and induces angiogenesis. Since at least one PPARd agonist is currently under clinical evaluation for treatment of dyslipidemias and obesity, our results raise serious concerns about the use of these agents in people at high risk for colorectal cancer. Our preliminary data also indicate that activation of PPARd inhibits the ability of PPAR? to induce tumor cell death in vitro. We postulate that inhibition of PPARd may restore the ability of PPAR? agonists to induce apoptosis in vivo. We have recently identified a nuclear receptor NR4A2 which is a novel PGE2-regulated gene. NR4A2 is a nuclear receptor known to be involved in regulating brain function. We found that NR4A2 is elevated in human colorectal cancers and mediates the PGE2-induced activation of PPARd transcriptional activity by directly interacting with PPARd. However, the mechanisms by which NR4A2 modulates PPARd transcriptional activity during colorectal carcinogenesis are not clear. We propose the following specific aims to investigate these problems: 1) investigate mechanisms by which PPARd accelerates intestinal polyp growth and characterize PPARd target genes that control tumor growth; 2) characterize the role of PPARd in modulating the antitumor effects of PPAR? in colorectal cancer; and 3) delineate the mechanisms by which other nuclear receptors such as NR4A2 regulate PPARd transactivation. The results of the experiments proposed here may lead to the design of novel approaches for prevention and/or treatment of colorectal cancer. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): This is a resubmission for The University of Puerto Rico Comprehensive Cancer Center (UPRCCC) and The University of Texas M. D. Anderson Cancer Center (MDACC) Partnership for Excellence in Research Grant, which addresses comments and recommendations from the 1) summary statement, 2) NCI Site Visit, and 3) IAC and PSC Committees. Areas strengthened in this application include: leadership, communication, committee oversight, evaluation, career development and mentorship. Funds granted in this award will be used to: 1) support 3 full projects in cancer biology and prevention and 1 pilot project in epidemiology, 2) enhance existing collaborations and establish new collaborations, 3) increase faculty/trainee participation in the training program, 4) expand efforts in our Outreach Program to impact a larger audience, and 5) fortify and stabilize the infrastructure developing at the UPRCCC. Research grants included target 1) developing new cancer imaging techniques, 2) regulating RNA in eukaryotic cells, 3) reducing cancer risk through combined alcohol and tobacco treatment and 4) assessing the role of insulin resistance syndrome as a risk factor for endometrial cancer. The Outreach Program, which was added into the original grant mid-year 3, has been expanded to include cancer related to tobacco, as well as breast, cervical and HPV. The Training Program has also been expanded in scope to impact more faculty and trainees through a comprehensive career development and mentorship training program. The original grant (2002) was awarded to establish a comprehensive long-term mutually beneficial relationship to target and eradicate cancer health disparities. The two short term objectives of the original application were met: 1) develop independent cancer research investigators and 2) establish a strong working relationship based on mutual benefit, identified strengths and reciprocity. The 3rd long-term objective of the Partnership is to regain NCI-designation for the UPRCCC. Thus far, the Partnership has set the foundation for a dynamic collaboration dedicated to enhancing each partner's strengths and eliminating each partner's weaknesses. The Partnership has also served as the catalyst for two major laws passed in Puerto Rico, one of which resulted in the allocation of funding for establishing the infrastructure and recruiting top notch faculty to the UPRCCC. As of 2008, the Partnership is far from reaching its long-range goal of regaining NCI-designation for the UPRCCC. Funding for the U54 resubmission is critical to maintain the momentum of the Partnership's joint efforts in Cancer Research, Training and Outreach. [unreadable] [unreadable] [unreadable] [unreadable]

Colorectal cancer affects a significant number of individuals in most industrialized countries around the world. It alone is responsible for an annual death toll in the United States of approximately 56,000 people. Many patients present with advanced disease with which the outcome is poor and mortality high even with the most advanced treatment options available. Based on a better understanding of the biology of the large intestine and the specific signaling pathways affected during colorectal carcinogenesis, investigators are evaluating better ways to detect and prevent this disease. Along these lines, we have found that a particular enzyme, cyclooxygenase-2 (COX-2), is upregulated in colorectal cancer tissue and in pre-malignant adenomas. Our previous funding cycle was devoted to understanding the mechanisms which led to increased expression of the COX-2 gene and the role this gene played in gut biology and carcinogenesis. Now we propose to evaluate the downstream signaling pathways affected by increased COX-2 activity. Cyclooxygenase is a key enzyme in the production of prostaglandins which are bioactive lipids that activate several downstream signaling pathways. Others have shown that overexpression of COX-2 in the skin or breast leads to a dramatic increase in risk for skin or breast carcinoma. We have shown in preliminary data that PGEz can lead to pro-neoplastic effects in a number of situations. We propose the following specific alms to investigate this problem further: 1) To determine the mechanism(s) by which COX-2 derived PGE2 regulates the motility and invasiveness of colorectal carcinoma cells. 2) To delineate the role of the nuclear PGI2 receptor, PPAR_i, in the development of coloreetal cancer. 3) To determine the relative contribution of COX-2 expressed in malignant epithelial cells versus the surrounding stroma (host) in promoting colorectal cancer. This work will help to delineate additional important targets for treatment and/or prevention of colorectal cancer. It is becoming clear that just targeting one single molecule will not be as effective as needed to have a significant impact on this disease.

This is a resubmission for The University of Puerto Rico Comprehensive Cancer Center (UPRCCC) and The University of Texas M. D. Anderson Cancer Center (MDACC) Partnership for Excellence in Research Grant, which addresses comments and recommendations from the 1) summary statement, 2) NCI Site Visit, and 3) IAC and PSC Committees. Areas strengthened in this application include: leadership, communication, committee oversight, evaluation, career development and mentorship. Funds granted in this award will be used to: 1) support 3 full projects in cancer biology and prevention and 1 pilot project in epidemiology, 2) enhance existing collaborations and establish new collaborations, 3) increase faculty/trainee participation in the training program, 4) expand efforts in our Outreach Program to impact a larger audience, and 5) fortify and stabilize the infrastructure developing at the UPRCCC. Research grants included target 1) developing new cancer imaging techniques, 2) regulating RNA in eukaryotic cells, 3) reducing cancer risk through combined alcohol and tobacco treatment and 4) assessing the role of insulin resistance syndrome as a risk factor for endometrial cancer. The Outreach Program, which was added into the original grant mid-year 3, has been expanded to include cancer related to tobacco, as well as breast, cervical and HPV. The Training Program has also been expanded in scope to impact more faculty and trainees through a comprehensive career development and mentorship training program. The original grant (2002) was awarded to establish a comprehensive long-term mutually beneficial relationship to target and eradicate cancer health disparities. The two short term objectives of the original proposal were met: 1) develop independent cancer research investigators and 2) establish a strong working relationship based on mutual benefit, identified strengths and reciprocity. The 3rd long-term objective of the Partnership is to regain NCI-designation for the UPRCCC. Thus far, the Partnership has set the foundation for a dynamic collaboration dedicated to enhancing each partner's strengths and eliminating each partner's weaknesses. The Partnership has also served as the catalyst for two major laws passed in Puerto Rico, one of which resulted in the allocation of funding for establishing the infrastructure and recruiting top notch faculty to the UPRCCC. As of 2008, the Partnership is far from reaching its long-range goal of regaining NCI-designation for the UPRCCC. Funding for the U54 resubmission is critical to maintain the momentum of the Partnership's joint efforts in Cancer Research, Training and Outreach.

Pro-inflammatory prostaglandin E2 (PGE2) is the most abundant prostaglandin found in human colorectal cancers and plays a predominant role in promoting tumor growth. In contrast to the role of PGE2 in carcinogenesis and inflammation, another of the arachidonate-based bioactive lipids, endocannabinoids, exert potential anti-tumor effects on a wide spectrum of human tumors in vitro and have proven antiinflammatory properties. Opposing effects of PGE2 and endocannabinoids on inflammation and cancer progression prompted us to hypothesize that PGE2 and endocannabinoids coordinately control tumor progression. Endocannabinoids primarily activate two G-protein-coupled cannabinoid receptors CBI and CB2 and are degraded by fatty acid amide hydrolase (FAAH). Based on our preliminary data, cross talk exists between PGE2 and endocannabinoid signaling. PGE2 can downregulate CBI expression via DNA methylation, whereas a selective COX-2 inhibitor restores CBI expression. However, little is known regarding the role of CB1, CB2 and FAAH in colorectal cancer progression. This proposal will examine the following: 1) The role of PGE2 in silencing tumor suppressors, DNA repair genes, and tumor inhibitory genes via DNA methylation during the colon tumor progression. 2) The role of endocannabinoid signaling in colorectal cancer progression by examining the biological functions of FAAH and by evaluating whether a combinational treatment of a CB1 agonist with a demethylating agent or a selective COX-2 inhibitor achieves optimal anti-tumor effects of CBI agonists in vivo. 3) The influence of endocannabinoid signaling on inflammation-associated colorectal tumorigenesis by defining the role of CB1, CB2 and FAAH in the mouse models of inflammation-associated colonic carcinogenesis and by evaluating the relative contributions of the endocannabinoid signaling in epithelial cells versus immune cells. In summary, we expect to generate novel mouse models and new approaches to study the role of PGE2-mediated inflammation, its influence on DNA methylation and suppression of the anti-inflammatory role of the endocannabinoid pathway in the progression of colorectal cancer.

DESCRIPTION (provided by applicant): Epidemiologic and clinical evidence has demonstrated that nonsteroidal anti-inflammatory drugs (NSAIDs), especially aspirin, protects against colorectal cancer (CRC) incidence and mortality. Elucidating the mechanism(s) responsible for the protective effect of NSAIDs could lead to major breakthroughs in the field of cancer chemoprevention and treatment. The most compelling evidence to date indicates that reduction of pro- inflammatory prostaglandin E2 (PGE2) production via inhibition of the cyclooxygenase pathway is responsible for part of this protective effect. Given that PGE2 can mediate the tumor-promoting effects of COX-1 and COX- 2, more selective pharmacological inhibition of PGE2 signaling may be efficacious in preventing CRC with avoiding the cardiovascular and other side effects associated with NSAID use. Although our group and others have extensively investigated the mechanisms by which PGE2 promotes CRC progression, it is still not fully understood how PGE2 accelerates CRC formation and progression. During the current funding period, our progress has led to many milestones in understanding the central role of PGE2 signaling in CRC and also provides the foundation for future investigation. Our preliminary data shows that PGE2 stimulates the expansion of colonic cancer stem cell (tumor-initiating cell) in vitro and in vivo. However, it is unclear whether PGE2 promotes CRC initiation and progression through enhancing cancer stem cell growth, maturation, and survival. We will address these questions in the Aim 1. Moreover, our preliminary results indicate that the CXCR2, one of downstream targets of PGE2, promotes chronic intestinal inflammation and colitis-associated tumorigenesis. We further provide the first evidence showing that CXCR2 is required to recruit myeloid-derived suppressor cells (MDSCs) to the colonic mucosal tissue. Previously, this receptor was thought to only mediate neutrophil migration to inflammatory sites. Although evidence for MDSC promotion of immunosuppression is accumulating, it remains unclear whether CXCR2-expressing MDSCs play a key role in connecting colonic chronic inflammation to colitis-associated carcinogenesis. Aim 2 is designed to address these questions. Finally, our preliminary data revealed that adoptive transfer of human natural killer (NK) cells significantly inhibited liver metastases in a mouse model of CRC metastasis. However, the role of NK cells in inhibiting metastatic CRC and preventing tumor recurrence after surgery remains unclear. In addition, we will evaluate whether inhibition of MDSC recruitment by CXCR2 antagonists and reduction of MDSC numbers in tumor by aspirin enhance the ability of NK cells to eliminate CRC liver metastases. We will address these issue in Aim 3. Collectively, the results from our renewal proposal will not only reveal comprehensive insights of how PGE2, CXCR2, MDSCs, and NK cells coordinately contribute to CRC initiation, progression, and metastasis, but also provide a rational for applying adoptive transfer of allogeneic NK cells with subverting tumor-induced immunosuppression as novel therapeutic approaches in metastatic and adjuvant therapies.

DESCRIPTION (provided by applicant): Elucidating the molecular mechanism(s) by which aspirin use reduces the risk and mortality of colorectal cancer (CRC) could lead to major breakthroughs in the field of cancer chemoprevention and treatment. The most compelling evidence to date indicates that the anti-tumor effects of aspirin and other NSAIDs are due to reduction of pro-inflammatory prostaglandin E2 (PGE2) production via inhibiting cyclooxygenase enzymatic activity. Our preliminary data supports this hypothesis by showing that aspirin reduced polyp numbers along with a decrease of prostaglandin production in tumors. However, no direct evidence has been developed that aspirin inhibits CRC initiation, progression, and metastasis by reduction of PGE2 production via targeting COX enzymes. In addition, previous studies have focused on the roles of NSAIDs in eliminating tumor epithelial cells and suppressing tumor-associated angiogenesis. Little is known about the impact of aspirin on CRC immune evasion. Our observations, never before reported, indicate that aspirin restores host immunosurveillance by inhibition of myeloid-derived suppressor cells (MDSCs) and PGE2 induces an infiltration of MDSCs into the intestinal tumor and mucosa via induction of CXCR2 ligand expression prompted us to postulate that aspirin might inhibit tumor initiation, progression, and metastasis by suppressing recruitment of MDSCs via targeting a novel COX-PGE2-CXCR2 pathway. Aim 1 is designed to test this hypothesis. The results from this aim could not only identify key mechanisms responsible for anti-tumor effects of aspirin, but also may provide a rationale for development of new therapeutic approaches to subvert APC mutation- and tumor-induced immunosuppression by using CXCR2 antagonists and/or CXCR2 neutralizing antibodies. Moreover, our preliminary studies revealed for the first time that primary tumor induced immunosuppression in pre-metastatic organs, whereas treatment with a COXIB attenuated the effects of the primary tumor on pre-metastatic niche formation in the liver. Thus, it is conceivable to hypothesize that aspirin inhibits metastasis by blocking the formation of pre-metastatic niches via targeting the COX-PGE2-CXCR2 pathway. Aim 2 is designed to examine this postulation. The results from this aim should provide a rationale for developing novel therapeutic approaches to inhibit CRC metastasis by blocking the pre-metastatic niche formation. Finally, one potential explanation for the cancer-preventive effects of aspirin could be due to inhibition of COX-1 activity in platelets. However, there is no clear evidence supporting this idea. We will test this role of platelet COX-1 in Aim 3. The results from this aim will provid a rationale for development of new therapeutic approaches to target platelet COX-1 in future cancer prevention and treatment efforts. Collectively, the mechanisms we identify in this proposal might be applicable for other solid cancers in general and can certainly be tested in other systems. In addition, targeting host immunosurveillance or platelets may also represent a novel therapeutic approach for CRC patients.

DESCRIPTION (provided by applicant): This proposal requests continued support of the grant T35 DK007431 for short-term research training experiences at the Medical University of South Carolina for medical students in areas that fall within the broad spectrum of NIDDK mission areas such as diabetes, metabolic diseases, endocrine disorders, as well as the basic biology and pathophysiology of kidney, digestive, urologic and hematologic diseases. The program provides a 10-12 week research project, under the supervision of an established investigator, accompanied by a set of structured enrichment activities. Tracking data for medical students trained over the past 10-year period indicate that 83% of them pursued residency positions at prestigious, academic medical centers where clinically relevant questions can be addressed through research studies. Our NIDDK short-term research training program exposes medical students and other health professionals to a diverse array of expertise, strategies, technologies and questions that underlie many of the most serious diseases affecting public health. The strengths of the program include the expertise of the mentors, basic and clinical scientists working side-by side, a vast array of technology and clinically relevant disease models for studying molecular events involved in disease processes, ability to ask significant questions about these events, and experience with and commitment to research training from bench to bedside. Our trainees will benefit from understanding the critical issues pertinent to preclinical development and implementation of new diagnostics and therapies. The Program Faculty represent foci of research excellence in five broad areas: Diabetes and Complications; Renal, Vascular and Hepato-Pathophysiology; Signal Transduction and Human Disease; Health Disparities and Gender Differences; New Technologies and Human Disease Research. All students are required to participate in a class entitled Responsible Conduct of Research, given the first week of the summer program, and in a course introducing them to NIDDK clinical research. Students present their research results at the end of the summer in a Summer Health Professionals (SHP) Colloquium, and again in either poster or oral format at MUSC's Student Research Day in November. Research electives in the medical school curriculum are available for students who wish to conduct a second short-term experience, generally in their fourth year of study. The Program is directed by an NIDDK-funded investigator with a medical background, clinical research orientation, and leadership in health disparities research. He will be assisted b a Steering Committee of 6 members representing the basic and clinical sciences. An evaluation plan that incorporates both quantitative and qualitative data, meaningful comparisons, and multiple sources of information will be used to guide and inform the program.