Alec J. Davidson - US grants

[unreadable] DESCRIPTION (provided by applicant): Circadian rhythmicity provides a critically important temporal framework for many molecular, cellular and organismal functions. Until recently it was thought that circadian rhythms in mammals were generated by a single 'master oscillator' in the brain, the SCN. We now know that virtually all cells and tissues have molecular machinery capable of generating circadian rhythms. Several recent reports suggest that experimental manipulation of host circadian rhythmicity affects tumor growth and mortality in rodents with cancer. In humans there are indications that altered circadian rhythmicity correlates with risk of cancer and with survival statistics in diagnosed patients. Therefore the role played by circadian rhythms in host and tumor cells in the development of cancer is of great interest. Restricted feeding schedules synchronize circadian rhythms in behavior, physiology and gene expression in digestive organs was also shown to inhibit growth of Glasgow osteosarcoma and to slow associated mortality in mice. Therefore we propose a project to develop a better understanding of the impact of restricted feeding schedules on cancer prevention and development and tumor rhythmicity in liver. We will develop a new model to study the role of molecular circadian rhythms in the ontogeny and progression of hepatocellular carcinoma (HCC). Rats bearing the reporter gene Per1-luciferase will be administered Diethylnitrosamine (DENA). We will then expose these rats to daytime, nighttime, or non-circadian restricted feeding schedules. Tumor development will be assessed once per month by small-bore MRI and by mortality. In parallel we will assess the effects of each nutritional manipulation on clock gene expression patterns in healthy liver and in liver tumors. These studies will test the hypothesis that rhythmicity in peripheral structures is intimately related to the organism's ability to combat the development and progression of cancer. The experiments will form the basis for future study of circadian and nutritional influences on development of this and other cancers. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The molecular machinery that governs circadian rhythmicity is comprised of transcriptional translational feedback loop whereby clock1 gene products inhibit their own transcription. Experimental manipulation of rhythms by brain lesions altered lighting environments and timed nutritional restriction can affect tumor growth and survival times in laboratory rodent models. It has been shown that aberrant circadian rhythmicity correlates with an increased risk of cancer development and with decreased survival statistics in diagnosed cancer patients. [unreadable] [unreadable] Using newly developed bioluminescent mouse tumor models, bioluminescent prostate cancer cell lines, [unreadable] and newly-developed in vivo molecular imaging techniques, we propose to illustrate the spatiotemporal [unreadable] dynamics of circadian rhythms of gene expression in healthy prostate and prostate cancer. We will also [unreadable] examine whether temporally restricted nutrition regulates prostate rhythmicity, and tumorigenesis and [unreadable] progression. [unreadable] [unreadable] Specific Aim 1: To evaluate whether prostate cancers have altered circadian rhythms of gene expression compared with healthy prostate tissue via in vitro and in vivo tracking of bioluminescent reporter genes. We will generate a dual transgenic mouse that develops prostate cancer and expresses the Per2:luciferase fusion protein and characterize the mPer2 gene temporal expression profile in prostate intraepithelial neoplasia (PIN), prostate adenocarcinoma, and metastatic foci and in healthy surrounding tissue. In parallel, we will develop a tumorigenic prostate cancer cell line, syngeneic on C57BL/6, which expresses circadian luciferase reporter genes to allow for in vivo tracking of tumor growth and molecular rhythms in tumors in anesthetized mice, and eventually in awake, behaving mice. [unreadable] [unreadable] Specific Aim 2: To test the hypothesis that nutritional manipulations of circadian rhythms differentially [unreadable] affect tumor development We will expose the dual transgenic mice or their WT littermates with bioluminescent tumors to nutritional manipulations known to alter molecular and physiological rhythmicity in peripheral organs: daytime restricted feeding, nighttime restricted feeding, and, as a control, caloric restriction without circadian entrainment. Tumor development and progression will be assessed longitudinally in mouse via in vivo imaging of tumor volume. In parallel, we will assess the effects of each circadian manipulation on circadian clock gene expression patterns in various stages of primary prostate cancer, in healthy prostate gland and in subcutaneous bioluminescent prostate adenocarcinoma cells. [unreadable] [unreadable] [unreadable] [unreadable]

Initial Planning Stage Drs. Chung and Davidson have already identified an area of common interest and of high importance: the circadian biology of prostate cancer. Further, we have selected an Internal Advisory Committee made up of 6 distinguished cancer faculty, 3 from each partner institution to help select the 2nd and any future collaborative pilot projects that are to be funded via this grant. Consequently, the Planning Stage is virtually complete. For this reason we are only requesting 3 years of support, making this section unnecessary for our application. The only planning activities proposed are a series of lectures occurring both at MSM and WCI that are intended to help foster collaborations beyond those already proposed in this application. We will hold 4 lectures, 2 at MSM where Emory faculty involved in cancer research will be invited to speak about their research and meet with trainees. AUC faculty and students will be invited to the lectures. The other two will be presented by MSM cancer faculty at Emory. We will also host one major lecture per year at MSM on the topic of circadian biology of cancer. The speaker will be an internationally recognized cancer scientist or physician.

Priority-Setting Stage Pilot Project #1 (Drs. Davidson and Hsieh) is fully conceived, planned and staffed. Consequently this project will be implemented immediately upon funding. Pilot project #2 will be selected during Year 1 by a Priority-setting process described as follows. We have selected an Internal Advisory Committee comprised of 6 cancer investigators: The two Lead Pis plus 2 additional members from each institution: Morehouse School of Medicine Alec J. Davidson, PhD is an assistant professor in the Department of Anatomy and Neurobiology, and a member of the Neuroscience Institute and Circadian Rhythms and Sleep Disorders Program. Dr. Davidson has been pursuing research on the relationship between circadian clocks and cancer for 4 years, and is currently PI of an R21 grant from NCI to investigate this issue in a rat model of hepatocellular carcinoma. He is the MSM Lead-Pi of this grant application. Sandra Harris-Hooker, PhD is the Vice President and Associate Dean for Sponsored Research Administration at MSM, and the Director of the Minority Biomedical Research Support Program at Morehouse School of Medicine, a program dedicated to the development of future biomedical research scholars. With professional training in Cell Biology, and postgraduate emphasis in Cardiovascular Pathology, Dr. Harris- Hooker has worked in the area of vascular cell biology for over 20 years, studying endothelial dysfunctions involved in the pathogenesis of atherosclerosis. While her primary research area is vascular biology, she is also interested in ways by which to enhance the integration of basic, clinical and population-based research in order to address disparities in health. Dr. Harris-Hooker has been on the MSM faculty since 1983. Daniel Blumenthal, MD, MPH, is Chairman of the Department of Community Health and Preventive Medicine and Principal Investigator of the Morehouse School of Medicine Prevention Research Center (PRC). The mission of the PRC is to advance scientific knowledge in the field of prevention of disease in African American and other minority communities and to disseminate new information and strategies of prevention. Specifically, Dr. Blumenthal is PI of the Cancer Prevention and Control Research network within the PRC, whose mission it is to accelerate the adoption of evidence-based cancer prevention and control in minority communities. Winship Cancer Institute / Emory University Leland W. K. Chung, PhD, is an internationally recognized basic and translational prostate cancer scientist with substantial experience in coordinating multi-dimensional research projects and is currently the PI of a NCI funded prostate cancer program project grant (P01). Dr. Chung is a Professor of Urology, Biochemistry, and Hematology &Oncology, Director of the Molecular Urology and Therapeutics Program, and Director of the Prostate Cancer Program, WCI. Dr. Chung is the WCI Lead-Pi of this grant application. Dr. Chung's research has continuously received funding from the National Institutes of Health and National Cancer Institute over the past 30 years. He currently serves as the Principal Investigator of one NCI Program Projects entitled "Prostate cancer bone metastasis: biology and targeting" (Total: $6,016,114), a DoD Synergy Consortium targeting prostate cancer lethal phenotypes (Total: $9,999,999), two DoD Idea Awards grants entitled "Beta-2 microglobulin: a novel therapeutic target for the treatment of human prostate cancer bone metastasis" (Total: $573,750) and "Molecular imaging with quantum dots probing EMT and prostate cancer metastasis in live animals" (Total $573,750), and two industrial grants entitled "CNTO95: targeting prostate cancer bone metastasis" (Total $100,000), "Assessment of the co-targeting potential of prostate cancer metastasis" (Total $100,000);Dr. Chung currently serves as a Co-Investigator for an NCI sponsored Cancer Nanotechnology Center grant (Total: $7,239,413, PI: Shuming Nie), a P20 grant ($2,653,181, PI: Shuming Nie), and an R01 grant ($7,094,005, PI: Shuming Nie). Otis W. Brawley, MD, is professor of hematology, oncology, and medicine at the Emory University School of Medicine and Professor of Epidemiology at the Emory Rollins School of Public Health. He also serves as associate director of the Winship Cancer Institute at Emory University. In addition, Dr. Brawley is chief of hematology and oncology services and the medical director of the Georgia Cancer Coalition Center of Excellence at Grady Memorial Hospital. From 1995 to April 2001, he served as Assistant Director of the Office of Special Populations Research at the National Cancer Institute. Dr. Brawley was previously a senior in the Division of Cancer Prevention and Control at the National Cancer Institute where he was one of a group instrumental in the development and launching of the Prostate Cancer Prevention Trial. This 18,000-man trial is looking at screening and epidemiologic issues in prostate cancer as well as the potential for prevention of benign prostatic hyperplasia and prostate cancer. He also served as Chief of the NCI Intramural Prostate Cancer Clinic from 1993-1995. Dr. Brawley's research interests include the screening, epidemiology, diagnosis, prevention, and treatment of hormonal cancer. He has additional interests in the design of clinical trials, inclusion of minorities in trials and the availability of state-of-the-art health care to the socioeconomically disadvantaged. His work concerning racial differences in patterns of medical care and the similar outcomes among racial and ethnic groups when there is equal treatment is widely cited in medical and lay literature. He is the recipient of numerous awards and was recently named a Georgia Cancer Coalition Eminent Scholar. Jin-Tang Dong, PhD, is currently an Associate Professor of Oncology, Urology, and Genetics and Molecular Biology at Emory University School of Medicine. His research has primarily focused on two areas of cancer biology - cancer gene identification and molecular understanding of two cancer molecules identified in his laboratory (ATBF1 and KLF5). He has published more than 50 research papers and trained more than 20 people of varying levels in cancer biology. He participates regularly in grant reviewing for NIH and US Army breast cancer and prostate cancer programs. Selection of Pilot Project 2 During the first quarter of Year 1, Drs. Davidson and Chung will write a Request for Applications that will be distributed by broadcast email and fliers within WCI and MSM. Potential applicants will be asked to conceive of projects that are bidirectionally collaborative (benefiting both institutions), of high scientific value, and investigate an area of cancer biology that might yield insight regarding racial and ethnic health disparities (preferably prostate cancer). This includes the cellular, molecular or systems biology of cancers that are more diagnosed, or more deadly among minority populations. Successful applications must conform to the stipulations published in PAR-07-230, the announcement for this parent application. Any applications from trainees or faculty that have not yet successfully obtained any independent research funding will require a career development plan. The administrative core will collect and duplicate (or electronically distribute) the applications as needed for review. If applications are not responsive to the RFA they will be returned to applicants with that indication. After the application deadline, the IAC will be provided with the responsive applications, and asked to assign them priority ranks based on the criteria of: scientific merit, the strength of the collaboration (must be mutually beneficial), the potential of the project to generate publishable data and a competitive independent grant application, and the training potential for trainees and junior faculty. If the committee lacks the scientific expertise to evaluate applications for specific projects/programs, ad hoc reviewers either from within or outside the partnering institutions may be added to the IAC. Once scores have been assigned the IAC will then meet briefly to confer on their scores and decide which project to fund. If pilot project budgets are significantly less than the $120,000 maximum, the committee may choose to fund more than one project. In addition to reviewing and prioritizing new pilot projects/programs, the IAC will also evaluate ongoing pilot projects/programs and advise the Pis from both partnering components as to the general activities of the partnership and how they may contribute effectively to achieving high-priority goals and objectives. In exchange for their yearly service, committee members (but not the Lead Pis) and ad hoc reviewers (if necessary) will be compensated as consultants ($300 per year) as part of the budget for the administrative core.

Address; Affect; Age; Aging; Animals; Behavior; Body Tissues; Brain; Cessation of life; Chronic; Circadian Rhythms; Common Rat Strains; Controlled Study; Coronary Disease; Coronary heart disease; Cyclicity; Data; Death; Digestive Diseases; Digestive System Diseases; Digestive System Disorders; Disease; Disorder; Disruption; Diurnal Rhythm; EEG; EMG; Electroencephalography; Electromyography; Encephalon; Encephalons; Environment; Exposure to; Health; Human; Human, General; Illumination; Length of Life; Light; Lighting; Longevity; Mammalia; Mammals; Mammals, General; Mammals, Mice; Mammals, Rats; Mammals, Rodents; Man (Taxonomy); Man, Modern; Medical; Mice; Minority; Molecular; Monitor; Monitoring, Sleep; Morbidity; Morbidity - disease rate; Mortality; Mortality Vital Statistics; Murine; Mus; Nervous System, Brain; Nyctohemeral Rhythm; Organ; Outcome; Pattern; Periodicity; Peripheral; Phase; Photoradiation; Play; Polysomnography; Rat; Rate; Rattus; Reporting; Rhythmicity; Risk; Rodent; Rodentia; Rodentias; Role; SCHED; Sampling; Schedule; Senescence; Simulate; Sleep; Sleep Deprivation; Sleep Disorders; Societies; Somnography; Standards; Standards of Weights and Measures; System; System, LOINC Axis 4; Testing; Time; Tissues; Travel; Twenty-Four Hour Rhythm; United States; Work; age dependent; age related; aged; cancer risk; circadian; circadian process; coronary disorder; daily biorhythm; day; day shift; digestive disorder; disease/disorder; diurnal variation; experience; indexing; juvenile animal; life span; lifespan; mid life; mid-life; middle age; middle aged; midlife; mouse model; night shift; night work; polysomnographic; senescent; shift work; sleep measurement; sleep polysomnography; sleep problem; social role; young animal

? DESCRIPTION (provided by applicant): Shift workers comprise nearly 20% of the US working population, and exhibit disparately high rates of several cancers, obesity, diabetes, stroke, and circulatory disease. A better understanding of the mechanistic factors contributing to increased morbidity in shift workers is needed before the risks associated with these work schedules can be reduced. Simulated rotating shift work in mice, termed Environmental Circadian Disruption (ECD), leads to sleep loss, accelerates death, and dysregulates innate immune function. ECD-exposed mice exhibit pathologically severe inflammatory responses to bacterial endotoxin and increased infarct size following ischemic stroke. Our preliminary work has helped us establish a hypothesis that ECD disrupts the immune cell molecular circadian clock and produces a novel form of immunosuppression. We posit that this immunosuppression specifically slows anti-inflammatory signaling during an inflammatory challenge due to loss of adequate signaling through the TAM family of receptor tyrosine kinases, resulting in uncontrolled pro- inflammatory cytokine release and pathology. In this grant we will investigate the mechanistic underpinnings of the immunological consequences of ECD using newly developed in vitro ECD paradigms, laying the groundwork for translation of our work to human subjects exposed to laboratory-simulated and real-world shift work. The long term goal of this research effort is to develop screening tools and preventative medical treatments that may mitigate the adverse health consequences associated with work schedules that cause circadian disruption.

PROJECT SUMMARY Due to the very recent development of in vivo widefield fluorescence imaging using a miniaturized mi- croscope, neuronal networks can now be observed in real time during responses to environmental input, goal- directed behaviors, and abnormal function with unprecedented anatomical and phenotypic specificity. The cir- cadian neural network in the hypothalamic suprachiasmatic nucleus (SCN) is an ideal model system in which to apply these tools. The SCN network is unique in that it receives direct monosynaptic sensory input from the eyes, encodes light onset and daylength into its network function, and provides neuronal output that regulates the timing of most physiological functions and behavior. However, a major limitation in investigations of SCN physiology to-date has been the need to section the brain, destroying most of the network, and divorcing it from its sensory inputs or behavioral outputs. The ability to study the intact SCN network with functional visual afferents and behavioral efferents, would represent an unprecedented leap forward for the field. We propose to develop a toolkit for the real-time imaging with cell-specific optogenetic control of SCN neurophysiological activity. This requires optimization via systematic testing of genetic constructs, viral vectors, imaging devices, mouse models, and surgical procedures. Once optimized, we will use these powerful approaches to study a critically important feature of circadian biology which was previously unaddressable: the network processing of light input that leads to shifts of the circadian behavioral rhythm.

Project summary / Abstract A fundamental property of neural systems is the capability for functional adaptation in response to the signals they re- ceive. Though much has been learned using existing approaches, a major limitation in investigations of neural circuits has been the need to section the brain, destroying most of the network, and divorcing it from its sensory inputs or behavioral outputs. The mammalian circadian system, including the hypothalamic suprachiasmatic nucleus (SCN), is one such cir- cuit, unique in that represents a well-defined population of cells that engage in a full suite of functions that typify an entire nervous system: the network is affected directly by sensory input which is easily manipulated, processes such input to alter its function, and generates outputs that directly impact behavior. The overall goal of our research program is to un- derstand how the brain processes sensory light input, integrates that input into the ongoing circadian behavioral/regulatory program, and generates outputs that regulate behavior and physiological activity. We propose that it is a process involving hierarchical plasticity, where sensory neurons in the eyes affect the internal clock time of primary oscillator neurons, which then act on secondary and tertiary systems within the brain to alter global physiology and the timing of sleep/wake. With in vivo widefield fluorescence imaging using a miniaturized microscope, this circadian neural network can now be observed in real time during responses to environmental input, goal-directed behaviors, and abnormal function. This ap- proach provides unprecedented anatomical and phenotypic specificity and can be applied over short (seconds-minutes) and long (weeks-months) timescales. Our research program comprehensively attacks a range of important systems-level features of circadian biology which were previously not addressable: cell-type specific acute response to systemic input (light), the on-going network level processing that occurs in response to that light input, and how those mechanisms begin to fail with aging. Such studies of hierarchical processing of sensory input, leading to systems-level plasticity and behav- ioral change, followed over long timescales will pay dividends in understanding the steady-state and aging mammalian nervous system as a whole.