Steven K. Clinton - US grants

Laboratory investigations and descriptive epidemiologic studies suggest a role for dietary fat in the etiology of colon, breast, and prostate cancer. However, many case-control and prospective studies have provided inconsistent results and the benefits to be derived from changes in dietary fat intake are intensely debated. Some of the conflicting data results from major limitations in the methodology used to quantitate human fat intake. Estimates of fat intake from food-frequency questionnaires, food diaries, and diet-interviews are frequently associated with errors of 30% or more. This project will focus upon the development of a simple molecular laboratory test to quantitate dietary fat intake. I will define a set of human genes whose expression is modulated by dietary fat concentration. This will be accomplished through the cloning of human homologues of mouse genes which show increased or decreased activity in response to diets high in fat. Several genes, such as LFG1 (Low Fat Gene 1), have been identified using subtraction hybridization cloning techniques with tissue from mice fed diets varying only in fat concentration. Once the human LFG-1, and additional fat-regulated genes have been sequenced, they will be compared to known DNA sequences in computerized data bases. This will determine: (1) whether we have isolated a novel fat regulated gene, (2) whether the gene has been sequenced but not known to be regulated by fat, or (3) has been sequenced land known to be regulated by diet. We will then develop competitive polymerase chain reaction (PCR) techniques for the precise quantitation of LFG1 expression, and other dietary fat regulated genes, in very small tissue samples such as a blood sample, rectal mucosal biopsy, or fine needle biopsy. Using PCR technology we will characterize the expression of LFG1 and other fat regulated genes in a variety of normal and malignant human cells and tissues including: (1) circulating blood monocytes, (2) biopsy specimens of normal and malignant breast, colon, and prostate, and (3) human cells cultured with differing concentrations of serum lipids. The above studies will provide the data necessary for selecting a panel of genes which can be tested in short term studies in human volunteers consuming diets varying in dietary fat concentration. It is our goal that these techniques will ultimately be utilized as quantitative markers for fat intake in geographic epidemiologic studies, migrant population studies, prospective and case control studies, and as an indicator of compliance with a low fat diet in cancer prevention trials.

Background. Prostate cancer has become the most frequently diagnosed malignancy in American men, particularly the African American population which experiences the highest rates in the world. It is probable that a number of dietary factors modulate risk. The Health Professionals Follow-Up Study has detected a significant reduction in prostate cancer risk with the consumption of tomato products. Several additional lines of preliminary evidence derived by our group support the hypothesis that tomato products may reduce risk. The biologically active substances in tomatoes that may influence the prostate are unknown. One possibility is the carotenoid lycopene. Higher serum concentrations of lycopene are associated with reduced prostrate cancer risk in the Physicians Health Study. Lycopene is the most common carotenoid in the human prostate and has antiproliferative effects on prostate cells in vitro. Hypothesis. We hypothesize that diets supplemented with tomato products or lycopene inhibit de novo prostate carcinogenesis, prostate tumor growth rates, and metastases in well characterized rodent models. Experimental Approach. The effects of diets supplemented with tomato powder, tomato lipophilic extract, or lycopene on prostate carcinogenesis will be evaluated in five models. (1). NMU/androgen-induced rat prostate carcinogenesis. (2). The growth rate of the well differentiated, slow growing and androgen sensitive Dunning R3327H prostate adenocarcinoma in rats. (3). Metastatic spread of the MatLyLu poorly differentiated and androgen independent prostate adenocarcinoma in rats. (4). The growth rate of the human poorly differentiated, androgen resistant, and p53 mutant PC-3 prostate adenocarcinoma in SCID mice. (5). The growth rate of the human, poorly differentiated, hormone dependent, and p53 normal LNCaP human prostate adenocarcinoma in SCID mice. Relevance. These studies will provide critical data for the understanding of prostate cancer etiology and prevention as well as the potential development of a new chemopreventive agent.

DESCRIPTION (provided by applicant): Rationale. Diets rich in tomato products and soy are hypothesized to reduce the risk of prostate cancer or perhaps, enhance the efficacy of prostate cancer therapy. Supporting data is derived from epidemiologic investigations, rodent models, and in vitro cell culture studies. Our research team has developed a processed food/nutriceutical combining lycopene-rich tomato juice with soy germ, with the goal of developing a standardized and easily consumed nutriceutical that can be employed in future large-scale, randomized, long-term intervention trials focusing upon prevention or treatment of prostate cancer. Hypothesis. We propose that a daily serving of a novel tomato-soy nutriceutical (OSU-TSJ-001) will cause a dose dependent increase in bioactive components (isoflavones, polyphenols, carotenoids) in the prostate, and alter biomarker patterns suggesting chemopreventive or therapeutic properties for prostate cancer. Experimental Approach. We will conduct a dose escalating phase I pre-prostatectomy clinical trial in 60 men that have clinically localized prostate cancer and have chosen prostatectomy for therapy. The dose escalating study will begin with 15 men consuming usual diets as the control group, with subsequent cohorts of 15 men consuming 5 oz per day, 10 oz per day, and 15 oz per day of the tomato-soy juice (OSU-TSJ-001). Men will consume the tomato-soy nutriceutical for a target of 28 days prior to undergoing prostatectomy. Aim 1. We hypothesize men will be compliant with consumption of the tomato-soy nutriceutical (OSU-TSJ- 001 ) with no dose limiting toxicity. History indicates that efforts to define toxicity and safety profiles are essential. Aim 2. We hypothesize that consumption of the tomato-soy nutriceutical (OSU-TSJ-001) will result in a dose dependent increase in the biodistribution of tomato-soy phytochemicals in blood and prostate tissue. Aim 3. We hypothesize that consumption of the tomato-soy nutriceutical (OSU-TSJ-001) alters blood hormonal patterns and biomarkers of oxidative stress that favor prostate cancer prevention. Aim 4. We will investigate histopathologic and molecular biomarkers associated with prostate carcinogenesis that may serve as surrogate endpoint biomarkers and provide information regarding their ability to be modulated by the tomato-soy nutriceutical (OSU-TSJ-001). Significance. The proposed study represents the first attempt to evaluate a novel "functional food" / neutriceutical containing tomato and soy components in a clinical trial targeting prostate cancer. Our objective is to develop the tomato-soy juice for future large scale randomized trials evaluating prostate cancer prevention or improved cancer therapy (prevent recurrence or improve outcomes of hormonal, biological, or chemical therapy). The phase I / II study provides essential safety and efficacy data along with valuable information regarding absorption and biodistribution of tomato-soy phytochemicals and the characterization of surrogate endpoint biomarkers relevant to mechanisms of action that may be useful in future randomized clinical trails.

DESCRIPTION (provided by applicant): Carotenoid-rich tomatoes and many products derived from tomatoes have been extensively marketed in the last decade for health promotion and disease prevention. Yet, controversy abounds, in large part because so little it known about the bioavailability, metabolism and mechanisms of action of the major tomato carotenoids lycopene (LYC), phytoene (PE) and phytofluene (PF). This grant application describes the characterization and implementation of a novel research tool that will dramatically improve our ability to trace the absorption of metabolites derived from the tomato carotenoids. The new technology, described and tested in this application, is based upon producing 13C labeled LYC, PE, and PF and testing them in humans. These 13C-enriched carotenoids will be used in a cross-over feeding study designed to more precisely define and compare how tomato carotenoids are absorbed and metabolized in men and women. Specific Aim 1: To produce pure 13C- phytoene (PE), 13C- phytofluene (PF) and 13C-lycopene (LYC) by utilizing in vitro tomato cell suspension culture technology. For the first time, we will apply tomato cell suspension culture for the reliable production of highly purified non-radioactive 13C-rich tomato carotenoids PE, PF, and LYC for novel bioavailability and metabolism studies in humans. Specific Aim #2: To compare and define kinetics of absorption and relative bioavailability of tomato-derived carotenoids in men and women using 13C -PE, 13C -PF, and 13C-LYC. We will use 13C-PE, 13C-PF, and 13C-LYC for characterization of uptake kinetics and relative bioavailability of these tomato carotenoids in healthy adult men (n=12) and women (n=12). Specific Aim 3: To identify metabolites of tomato-derived carotenoids in humans over time by employing 13C-PE, 13C-PF, or 13C-LYC. Knowledge and understanding of tomato-derived carotenoid metabolism in humans is speculative at best and essentially nonexistent with regards to both degradation pathways, and production of potentially bioactive compounds. Tracer labeling will allow us to examine the conversion of the orally administered parent compound to metabolites that are characteristic of bioactivation pathways or catabolic/degradation pathways using HPLC-MS/MS. Significance. Only through the development of novel tools to examine the digestion, absorption, bioavailability, tissue distribution, and systemic vs. target tissue metabolism will we be able to understand the mechanisms of action for LYC, PE, and PF in regards to human health. This proposal targets the Program Announcement PAR-08-135 entitled Exploratory/Developmental Grant for Complementary and Alternative Medicine (CAM) Studies of Humans of The National Center for Complementary and Alternative Medicine (NCCAM), NCI, the National Institute on Aging (NIA), and the Office of Dietary Supplements (ODS). PUBLIC HEALTH RELEVANCE: This proposal describes studies that characterize new techniques that will help us understand the absorption and metabolism of the major tomato carotenoids. These studies may help us defined the mechanisms of action for lycopene, phytofluene, and phytoene with regards to human health promotion and disease prevention.

DESCRIPTION (provided by applicant): Oral cancer affects nearly 300,000 Americans each year and is a serious health concern due to its poor prognosis and high recurrence rate. Moreover, the worldwide tobacco epidemic is leading to thousands of new cases of oral cancer being diagnosed each year. Targeting preventive strategies for those at highest risk for oral cancer due to tobacco exposure is thus a high priority. Our ultimate goal is to employ a food-based cancer preventive strategy for high-risk populations that allows excellent long-term adherence and efficacy. The oral cavity is an open microbial ecosystem that plays host to over 700 species of bacteria that form health- compatible communities called biofilms. We have previously demonstrated that these biofilms are rapidly enriched for pathogenic bacteria in smokers, resulting in an early hyper-inflammatory response. Furthermore, smoking cessation reverses this pathogenic bacterial recolonization, demonstrating that smoking has a direct effect on the oral microbiome and may increase the risk for oral carcinogenic processes by disturbing normal host-bacterial interactions. Therefore, our central hypothesis is that a critical bi-directional interaction exists between oral bacteria and phytochemical-rich black raspberry food products, which ultimately results in attenuation of inflammation and amelioration of disease. First, we will determine the effect of black raspberry phytochemicals (BRBs) on community dynamics within oral biofilms by combining a longitudinal clinical study design and a novel BRB delivery system (BRB nectar) with the resolution provided by shotgun metagenomic sequencing and computational bioinformatics. Next, we will examine the effect of oral bacterial communities on metabolism of BRBs in current and never smokers. Targeted HPLC-MS/MS analysis will be used to identify known phenolics and metabolites, while untargeted UHPLC Q-TOF metabolome analysis will identify novel compounds. Finally, we will evaluate the efficacy of BRBs and their metabolites in reversing the effect of smoking on oral host-microbial interactions by combining a longitudinal clinical study design with a novel, high-throughput dual RNA-Seq methodology to simultaneously quantify both oral bacterial and oral mucosal transcriptome changes before and after exposure to BRB nectar. Following the successful completion of these Specific Aims, we will demonstrate for the first time the ability of BRBs to mitigate pathogenic metabolic process and gene expression patterns induced by cigarette smoke in a manner that supports oral carcinogenesis prevention strategies.

DESCRIPTION (provided by applicant): The objective of this proposed Ohio State University (OSU) postdoctoral clinical T32 training program entitled: Training Hematology and Oncology Fellows in Clinical Research is to provide a state of the art clinical research training experienc specifically for postdoctoral fellows in hematology and oncology with an M.D., D.O. or equivalent degree who have demonstrated a firm commitment to patient oriented cancer research. The overriding goal of this training program is to prepare Hematology and Oncology fellows in the OSU Divisions of Hematology and Medical Oncology for academic careers in clinical and translational cancer investigation. The rationale for this training program is based on our firm belief that progress in cancer therapy and prevention will require strong collaborative efforts, and that patient oriented physician investigators will provide a critical link with physician-scientists and basic scientists toward this goal. The OSU Comprehensive Cancer Center (OSU CCC) provides a rich research environment that fosters collaborations among multiple disciplines, yet which currently lacks a focused mentoring experience for physicians interested primarily in pursuing patient oriented cancer research. The recent explosive growth of scientific discoveries and the increasing complexity of cancer clinical trials particularly, phase I/II studie of novel therapeutic agents and regimens, make it imperative that trainees in hematology and oncology acquire the proper mentoring and training to prepare them for careers in clinical and translational investigation. This program recognizes that successful preparation of fellowship trainees for productive academic careers is dependent upon many factors including an intellectually rigorous curriculum, adequate mentoring and support from senior faculty, protected time for academic training during fellowship, hands on experience in developing and writing clinical studies and research papers, and adequate opportunities to enhance their OSU experience with extramural programs and workshops sponsored by the NCI or other professional academic organizations such as ASCO and ASH. The participating members consist of the Program Directors, Coordinating Committee members, Advisory Committee members, primary mentors, and contributing faculty chosen based on their prior track record of successful mentoring as well as their demonstrated commitment to clinical and translational research. This program has a proposed duration of two years and will focus on the strengths of the OSU CCC, primarily in the fields of cancer prevention and experimental therapeutics. It is designed to foster and ensure the development of fellowship trainees who will be well prepared not only to assume positions as faculty members upon graduation but who will be empowered to become independent clinical and translational investigators in their own right. Furthermore, OSU is deeply committed to training women and members of underrepresented racial and ethnic groups for academic careers, and this program will build upon our proven success in recruiting and training female and underrepresented minority fellows.

PROJECT-004: MOLECULAR CARCINOGENESIS AND CHEMOPREVENTION PROGRAM (MCC) PROJECT SUMMARY / ABSTRACT The Molecular Carcinogenesis and Chemoprevention (MCC) Program, led by Steven K. Clinton, MD, PhD, has a collaborative team of 37 basic, translational and clinical scientists. These faculty have appointments in 17 Departments/Divisions within the Colleges of Medicine, Arts and Sciences, Pharmacy, Food Agriculture and Environmental Sciences, Public Health, Dentistry, Education and Human Ecology and Veterinary Medicine. The Specific Aims of the MCC Program are: 1) to characterize molecular and cellular changes induced by chemical, physical, hormonal and infectious agents that contribute to neoplastic transformation and multistage carcinogenesis in experimental models and humans; 2) to develop and characterize novel agents for cancer chemoprevention and define their efficacy, safety, and mechanisms of action using in vitro and preclinical models; and 3) to identify dietary and nutritional components that may enhance or inhibit the carcinogenesis cascade across the continuum of cancer progression. Each of these aims results in translational prevention studies in human populations with an emphasis on those at risk due to exposure to carcinogenic or cancer promoting agents, familial and genetic predisposition, or due to the presence of premalignant lesions. The MCC Program's overarching goals, implemented through multiple MCC initiatives, are to accelerate the research objectives of each Aim through incentivizing and stimulating collaborative investigation among MCC members, other investigators of the OSUCCC as well as facilitating the implementation of translational studies of cancer etiology, prevention, and progression in human trials. The MCC enhances quality by promoting knowledge of and utilization of state-of-the-art technologies provided by the OSUCCC shared resources (members utilize 14/14 shared resources). The MCC Program, during its previous review (2004-2009) was graded as ?Outstanding to Exceptional?. During this funding period (2009-2014), MCC Program members published 484 cancer relevant peer-reviewed articles in top tier journals for the respective fields of carcinogenesis, chemoprevention, and nutrition. Collaboration is extensive with 28% intra-programmatic publications and 55% inter-programmatic publications, with 272 or 56% being multi-institutional and 447 or 92% being collaborative publications. Peer-reviewed funding for the MCC Program is $5.19M in annual direct costs with $2.9M (56%) from the NCI. Translational research has been robust as well with 20 human trials led by MCC members employing the OSUCCC Clinical Trials Office resulting in 360 interventional accruals during the last funding cycle, 72% of which were from investigator-initiated Phase I and II trials. The current MCC Program uniquely integrates investigators across disciplines yet with shared interests focusing upon the interactive themes of carcinogenesis, chemoprevention, and nutrition. Our future goals include the integration of new initiatives involving the microbiome and metabolomics, two areas benefiting from rapid growth in technology and bioinformatics that will dramatically impact our understanding of carcinogenesis and strategies for cancer prevention.

DESCRIPTION (provided by applicant): Soybeans and the foods derived from them are a rich source of bioactive phytochemicals that promote health. Soy isoflavones are hypothesized to act through various pathways to impact prostate cancer (PCa) progression; however, one area that has received very little focus is the impact of soy components on the immune system. This is of importance with the ongoing development and approval of immune-based therapies for PCa (e.g.; Provenge) and a recent appreciation for the immune system in regulating prostate cancer development. One barrier to optimal anti-tumor immune responses are immunosuppressive myeloid-derived suppressor cells (MDSC). MDSC act through a variety of mechanisms systemically and in the tumor microenvironment. Our group and others have shown that MDSC are a relevant biomarker associated with reduced overall survival in patients with solid tumors and early reports indicate MDSC are elevated in PCa patients. Our preliminary data demonstrate that dietary soy modulates T cell proliferation and the cellular response to pro-inflammatory cytokines in mice. Further, genestein promoted in vitro differentiation of peripheral blood mononuclear cells into neutrophils and inhibited in vitro MDSC generation. We developed a novel soy bread which delivers high levels of isoflavones. This bread was safe and met with favorable compliance in men with PCa experiencing biochemical failure. Compared to baseline, MDSC and pro- inflammatory cytokines were significantly lower in the blood of patients receiving soy bread intervention. These data suggest that the role of dietary soy as an immunomodulator requires further systematic evaluation. We are interested in how dietary soy and its isoflavone constituents modulate signal transduction pathways and immune cell populations relevant to inflammation and prostate cancer. We hypothesize that dietary soy may prevent PCa progression by inhibiting expansion of immunosuppressive cells and reducing the cellular response to pro-inflammatory signals. We will determine how soy isoflavones or their metabolites modulate the differentiation and immunosuppressive function of myeloid cells after in vitro stimulation with pro- inflammatory stimuli. We will also evaluate the effects of dietary sy on MDSC expansion and function in the TRAMP and Hi-myc models of PCa and the ex vivo response of lymphocytes to relevant immune stimuli. A phase I clinical trial will be conducted in men diagnosed with PCa and randomized to receive dietary intervention with soy bread (experimental arm) or wheat bread (control arm) for 4 weeks prior to prostatectomy to evaluate pro-inflammatory cytokines, immune cell markers (e.g. MDSC, T cells, NK cells) and T cell proliferation in peripheral blood at baseline and following intervention and freshly isolated prostate tissue. These studies will provide a comprehensive, translational understanding of how dietary soy and its isoflavone components modulate aspects of immunity relevant to prostate cancer development and progression.

Project Summary/Abstract The evolution of prostate cancer from an androgen-dependent state (ADPC) to castration-resistant adenocarcinoma (CRPC) marks the lethal progression of the disease. Contemporary therapy for CRPC employs inhibitors of intra-tumoral and adrenal androgen synthesis (e.g. abiraterone acetate) or more potent androgen receptor (AR) antagonists (e.g. enzalutamide). However, these agents only provide a temporary response and modest increase in survival indicating a rapid evolution of resistance. In addition to CRPC, a significant number of patients develop small cell neuroendocrine carcinoma (SCNC) after hormonal therapy. SCNC is extremely aggressive and rapidly fatal. Importantly, with the widespread use of abiraterone acetate and enzalutamide, a greater frequency of the SCNC has been observed. Currently, there is no standard therapy that is effective for SCNC. Thus understanding the pathogenesis of CRPC/SCNC evolution and development of novel targeted therapies remain urgent needs. In addition, predictive and prognostic biomarkers that can help ?personalize? therapy and improve the precision of clinical trials are necessary. In preliminary studies, we have found that AKT-induced aberrant phosphorylation of transcription coactivator Mediator 1 (MED1) is required for UBE2C oncogene expression and for growth of CRPC and/or SCNC cells in vitro and in vivo. Furthermore, pharmacological AKT inhibition decreases UBE2C oncogene expression and cell growth in vitro in a phosphorylated MED1 (p-MED1)-specific manner. Importantly, the expression of p- MED1 significantly increases when human prostate cancer progresses to CRPC and SCNC. These findings support our hypothesis that AKT-induced aberrant MED1 phosphorylation drives an oncogenic gene expression program for CRPC/SCNC growth and progression. Our specific aims are to: (1) delineate the genomic mechanisms of p-MED1 binding to chromatin and global gene regulation in CRPC/SCNC; and (2) determine the biological impact and clinical relevance of AKT-p-MED1 transcriptional regulation in CRPC/SCNC. The successful completion of these aims will significantly impact the understanding of the critical oncogenic role of p-MED1 in CRPC and SCNC, laying the foundation for: (a) developing new therapies targeting the AKT-p-MED1 transcription axis, (b) employing p-MED1 as a marker for selecting AKT inhibitor- sensitive patients for ?precision medicine? clinical trials, and (c) identifying new predictive and prognostic biomarkers for CRPC and SCNC.

DESCRIPTION (provided by applicant): This project is based on the PI's novel finding that ?-tocopherol and, to a greater extent, ?-tocopherol facilitate the selective dephosphorylation of Akt at Ser-473 through PH domain-mediated membrane co- localization of Akt and PHLPP1 (PH domain leucine-rich repeat phosphatase 1). PHLPP1, a Ser-473 Akt phosphatase, acts as a tumor suppressor by negatively regulating Akt. From a mechanistic perspective, these findings provide the first evidence that ?-/?-tocopherol mediates redox-independent antitumor effects, at least in part, by counteracting the effect of phosphatidylinositol 3,4,5-trisphosphate on Akt activation. This unique mechanism provides a paradigm shift with respect to the regulation of Akt activity through membrane recruitment of PHLPP1, sheds light onto the enigma of how vitamin E mediates its chemopreventive effect and of why ?-tocopherol is more potent relative to the ? counterpart in suppressing cancer cell proliferation. In light of the tumor suppressor role of PHLPP in blocking PTEN mutant prostate carcinogenesis, this finding provides a molecular rationale for the use of ?-tocopherol as a scaffold to develop a novel class of PHLPP1-targeted Akt inhibitors, which have a distinct mode of action from other types of Akt inhibitors. The proof-of-concept of this lead optimization is provided by ?-VE5, a side chain-truncated ?-tocopherol derivative, which exhibited at least 20-fold higher potency relative to ?-tocopherol in mediating Akt dephosphorylation and growth inhibition of prostate cancer cells. Equally important, ?-VE5 exhibited in vivo efficacy in suppressing the growth of PTEN- deficient PC-3 and LNCaP-abl xenograft tumors in nude mice. Thus, this proposal consists of three specific aims with the goal of translating this novel mechanistic finding into a novel class of PHLPP1-targeted Akt inhibitors to block or delay the onset of prostate tumorigenesis. Aim 1 is to conduct structure-based lead optimization of ?-VE5 to develop more potent PHLPP1-targeted Akt inhibitors. Based on modeling and mutational analyses, we hypothesize that increasing polar interactions of the ligand with the hydrophilic residues in the binding pocket will enhance binding affinity for the PH domain. Proof-of-concept of this premise has been established by analysis of lead ?-VE5 derivatives. Continued optimization of these leads to generate 2nd generation compounds via isosteric replacement of metabolically labile moieties is proposed. Aim 2 is to investigate the mechanisms by which optimized ?-VE5 derivatives inhibit cell proliferation of PTEN-deficient prostate cancer cells. The top 3 optimal ?-VE5 derivatives from Aim 1 will be mechanistically validated by examining their effects on the activation status of Akt and various Akt downstream targets relevant to prostate carcinogenesis and tumor progression, especially glycogen synthase kinase (GSK)3ß, the forkhead box transcription factor Foxo3a, NF-?B, and AR signaling. As PHLPP1 plays a pivotal role in mediating the effect of AR inhibition on Akt activation in PTEN- deficient prostate cancer cells, the effects of these compounds on crosstalk of AR signaling with PHLPP1- mediated regulation of Akt activation will also be explored. In vivo efficacy of three optimal ?-VE5 derivatives will be evaluated in both PTEN-deficient (LNCaP-abl and PC-3) and PTEN-functional (22RV1) xenograft tumor models, which will be correlated with changes in the aforementioned biomarkers in tumors. Aim 3 is to assess the in vivo chemopreventive efficacy of a structurally optimized ?-VE5 derivative to block prostate tumorigenesis in the PTEN-knockout and TRAMP models. In light of the role of aberrant Akt signaling in prostate carcinogenesis, these two transgenic animal models represent therapeutically relevant models to evaluate the chemopreventive activities of these ?-VE5-derived PHLPP1-targeted Akt inhibitors.

Summary: Oral cancer affects nearly 300,000 Americans each year and is a serious health concern due to its poor prognosis and high recurrence rate. Moreover, the worldwide tobacco epidemic is leading to thousands of new cases of oral cancer being diagnosed each year. Targeting preventive strategies for those at highest risk for oral cancer due to tobacco exposure is thus a high priority. Our ultimate goal is to employ a food-based cancer preventive strategy for high-risk populations that allows excellent long-term adherence and efficacy. The oral cavity is an open microbial ecosystem that plays host to over 700 species of bacteria that form health compatible communities called biofilms. We have previously demonstrated that these biofilms are rapidly enriched for pathogenic bacteria in smokers, resulting in an early hyper-inflammatory response. Furthermore, smoking cessation reverses this pathogenic bacterial recolonization, demonstrating that smoking has a direct effect on the oral microbiome and may increase the risk for oral carcinogenic processes by disturbing normal host-bacterial interactions. Therefore, our central hypothesis is that a critical bi-directional interaction exists between oral bacteria and phytochemical-rich black raspberry food products, which ultimately results in attenuation of inflammation and amelioration of disease. The proposed Administrative Supplement is in response to PA-17-447: Validation Studies of Analytical Methods for Dietary Supplements and Natural Products. We aim to validate a UHPLC UV-Vis/QTOF MS method for quantifying urolithins and protocatechuic acid in biological specimens. Successful completion of this aim will specifically support a primary objective of U01CA188250 ?Interactive Omics: Black raspberry metabolites and the oral microbiome in smokers?. The aim is designed to examine the effect of oral bacterial communities on metabolism of BRBs in current and never smokers. The validated method will focus upon the targeted HPLC-MS/MS analysis will be used to identify known phenolics and metabolites. The validated method will be disseminated through publication and will support reproducibility in future research of putative biologically active botanical constituents, indicators of bioavailability, and of potential metabolic states.

PROJECT SUMMARY ? MOLECULAR CARCINOGENESIS AND CHEMOPREVENTION (MCC) The Molecular Carcinogenesis and Chemoprevention (MCC) Program at The Ohio State University Comprehensive Cancer Center (OSUCCC), led by Steven Clinton, MD, PhD, and Richard Fishel, PhD, has 48 members from 22 Departments and 8 OSU Colleges (Arts and Sciences, Dentistry, Education and Human Ecology, Food, Agriculture and Environmental Science, Medicine, Pharmacy, Public Health, and Veterinary Medicine). The MCC Program examines cancer as an integrated and dynamic process over time, with a major focus on the interface between genetics and the environment that collectively impacts the carcinogenesis cascade. This approach provides a foundation for chemoprevention and dietary interventions to reduce the burden of cancer in high-risk individuals as well as to decrease the incidence, mortality and morbidity of cancer in our Ohio catchment area (CA) and the nation. Our expertise extends to defining standards for genetic screening and counselling as well defining dietary and nutritional guidelines that impact cancer risk through public policy, nationally and globally. The Specific Aims of the MCC Program are: 1) Carcinogenesis: To characterize the genetic, molecular and cellular changes induced by germline, chemical, physical, hormonal or microbiological mediators that contribute to neoplastic transformation and multistage carcinogenesis; 2) Chemoprevention: To develop and characterize novel cancer chemopreventive agents and define their efficacy, safety, and mechanisms of action using biochemical, cellular and preclinical models that ultimately lead to early phase human studies; and, 3) Diet, Nutrition and Metabolism: To identify dietary patterns, nutritional components, and lifestyle variables that enhance or inhibit the carcinogenesis cascade across the continuum of cancer progression. MCC Program members published 547 cancer-relevant manuscripts between 12/01/14 and 11/30/19. Of these, 14% were intra- programmatic (multiple authors from MCC Program), 31% were inter-programmatic (authors from multiple OSUCCC Programs), and 72% were multi-institutional (authors from both CC and another institution). The total collaborative publications is 83%. MCC Program funding stands at $7.9M in overall direct, cancer-focused funding, of which $7.0M is peer-reviewed, including $6.8M direct funding from NIH ($3.4M from NCI). Over the last five years, MCC Program members have accrued 1,487 participants to trials; 670 to interventional trials and 817 to non-interventional trials. MCC members serve as leaders of the Ohio Colorectal Cancer Prevention Initiative involving 3,651 participants (3,310 colorectal and 341 endometrial) and ORIEN Total Cancer Care with enrollment of 50,683.The MCC program is fully integrated with the high priority crosscutting research initiatives of the OSUCCC and future plans complement and enhance programmatic aims while promoting interactions with the other research Programs and focus on 1) metabolic signatures in carcinogenesis and prevention; 2) the microbiome and immunology interface with Pelotonia Institute for Immuno-Oncology initiatives; and 3) collaborative efforts with the Center for Cancer Engineering.

PROJECT SUMMARY ? NUTRIENT AND PHYTOCHEMICAL ANALYTICS SHARED RESOURCE (NPASR) The mission of the NPASR is to provide niche services supporting research with high quality assays for biomarkers of tobacco exposure, specific nutrients, or a vast array of anti-cancer phytochemicals found in food sources. NPASR primarily supports the Molecular Carcinogenesis and Chemoprevention (MCC) and the Cancer Control (CC) Programs, which have robust research examining the role of diet and tobacco in cancer etiology, prevention, and survivorship. The NPASR co-Directors are Drs. Steven Clinton, a translational researcher in cancer prevention (interim co-Director, Senior Faculty Advisor; MCC co-Leader) and Devin Peterson, a senior food science chemist in the College of Food, Agricultural, and Environmental Sciences. The Specific Aims of the NPASR are to: 1) provide expert, leading-edge bioanalytical method development and quantitative analysis of nutrients and bioactive phytochemicals in foodstuffs; 2) conduct targeted quantitative analysis of nutrients, bioactive phytochemicals and their metabolites in biological samples generated from in vitro, in vivo and human studies using HPLC-MS/MS techniques; and 3) perform untargeted metabolomics and lipidomics services for cancer-related studies. NPASR technologies include state-of-the-art ultra-high performance liquid chromatography (UHPLC), mass spectrometry (MS) and liquid chromatography triple quadrupole MS/MS (LC- MS/MS). NPASR has added several new analytical capabilities during the current funding cycle including: 1) ion mobility hardware facilitating a broad semi-quantitative lipidomics platform; 2) a second MS instrument to support untargeted metabolomics and lipidomics demand; and 3) a state-of-the-art MS/MS for utmost sensitivity. During the current funding cycle, the NPASR supported 49 publications (2 > 10 impact factor), 55 users, and 6 NCI grants, including 1 P50, 3 R01s, 1 U19 and 1 U01, involving all five CCC programs. These efforts and publications have contributed to defining biomarkers of exposure or intake, and provided novel insight into phytochemical absorption, distribution, metabolism, and excretion. More recently, NPASR has expanded into targeted and untargeted metabolomics supporting our understanding of individual cancer risk due to tobacco exposure, nutrients, specific foods, and dietary patterns. To meet future demands of OSUCC Investigators aligned with OSUCCC strategic priorities, NPASR will further support, for example, studies of metabolomic- microbiome interactions, genetic determinants of metabolism and tobacco use biomarkers. During the next grant cycle, lipidomics and bioactive lipid analytic capabilities will be expanded to meet the needs of a growing user base of all five programs. The NPASR provides a critical service for CCC members evaluating foods, nutrients and carcinogens that spans research disciplines from cell culture and experimental animal studies to human trials and molecular epidemiology. The annual budget of the NPASR is $387,249, yet the CCSG request is $74,239. As such, the NPASR leverages extensive institutional support and seeks only 19.2% support from CCSG funds.