David Turner - US grants

Genetic and molecular studies have shown that proteins from the basic-helix-loop-helix (bHLH) family of transcription factors are key regulators of neural precursor formation and neuronal differentiation in vertebrates. These proteins include MASH1, XASH3, and members of the neuroD/MATH/neurogenin families. In Xenopus embryos, forced expression of a single bHLH transcription factor (e.g. neuroD1) can initiate neuronal differentiation, even in cells which are normally not destined to become neurons. In mice, targeted mutations in several of these genes have shown that they are essential for the formation of subsets of neurons. However, the molecular mechanisms by which the neural bHLH proteins function to initiate neuron formation or differentiation remain mostly unknown. To further our understanding of neural bHLH function, we propose to: 1) identify and characterize the domains of the neural bHLH proteins that are necessary for the formation of neurons, 2) characterize the activation of specific target genes by different neural bHLH proteins, and 3) analyze the inhibition of neural bHLH protein function by the Notch and ras signaling pathways. These studies will be facilitated by a mammalian cell culture model that we recently have developed. In this system, transfection of a single neural bHLH cDNA is sufficient to direct neuron formation from a multipotential cell line. This work should lead to a better understanding of the mechanisms that regulate normal neurogenesis and neuronal differentiation in mammals, including humans. In the long term, such information may contribute to developing strategies for replacement of neurons lost due to injury or neurodegenerative diseases.

[unreadable] DESCRIPTION (provided by applicant): The mammalian retina is composed of a diverse mixture of cell types with distinct functional roles (e.g. photoreceptors and multiple types of interneurons). The properties of distinct retinal cell types appear to be specified by different underlying patterns of gene expression, but the mechanisms that establish and maintain specific patterns of gene expression in individual retinal cell types are only partially understood. The recent discovery of an abundant class of small RNA regulatory molecules in animals, microRNAs (miRNAs), provides a potential novel regulatory mechanism for retinal cell diversification. Although several hundred miRNAs have been found to be expressed in the mammalian nervous system, recent computational searches for genes that encode miRNAs, as well as sequencing of miRNAs expressed in mammalian brain, suggest that numerous mammalian miRNAs remain to be identified. Genetic studies in the nematode C. elegans have identified miRNAs that control the identity of specific neurons and some of these miRNAs are expressed in very small populations of cells. If similar miRNAs, restricted to specific cell types, exist in the mammalian retina, those miRNAs from less abundant cell types will represent a small fraction of all miRNAs and therefore are unlikely to have been identified to date. [unreadable] [unreadable] The specific goals of the proposed project are to create an extensive profile of miRNAs expressed in the adult and neonatal mouse retina, to identify miRNAs that are restricted to specific retinal cell types or subtypes, and to identify retinal miRNAs with altered expression in the retinal degeneration 1 (Pde6brd1) mouse mutant, a model of human retinitis pigmentosa. miRNAs will be identified by extensive or deep sequencing, using a massively parallel sequencing technology. Several million short RNAs from adult and neonatal wild-type retinas and from adult Pde6brd1 retinas will be sequenced (>1000-fold deeper sequencing than prior analyses of retinal miRNAs). Sequences will be computationally analyzed to identify known and novel miRNAs and to exclude other RNAs. The miRNAs identified in the retina will be further characterized using recently developed miRNA in situ hybridization techniques. Based upon the frequency of different cell types in the retina, the deep sequencing of retinal miRNAs should allow the identification of miRNAs restricted to low abundance retinal cell types, as well as the generation of a comprehensive profile of miRNAs expressed in the retina. The proposed studies are intended to provide a framework for understanding miRNA expression and function in the mammalian retina. Identification of cell-type specific and other miRNAs in the retina will provide new genes that can be considered as candidates for human retinal diseases. In addition, expression of cell-type specific miRNAs may be subject to modulation in retinal diseases. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Post-transcriptional regulation of mRNAs by microRNAs is a widespread and essential component of gene regulation in animals. microRNAs, in association with Argonaute (Ago) proteins, bind to specific sequences in mRNAs to repress translation and/or reduce the level of the target mRNAs. Individual microRNAs may regulate many different genes, while individual mRNAs can be targeted by multiple microRNAs. Numerous microRNAs are expressed in the mammalian central nervous system (CNS), and microRNAs have been implicated in the regulation of a variety of processes in the CNS, including neurogenesis, cell survival and death, cell fate determination, neuronal differentiation, axon outgrowth, and synaptic function. Signal transduction plays essential roles in CNS development and function. Intracellular kinase cascades, activated by extracellular signals, directly modulate most biological processes, including transcription and translation. However, it is not known whether repression of target mRNAs by Ago/microRNA complexes can be modulated directly by signaling pathways. Phosphorylation of the Ago-2 protein by the p38 MAP kinase/MAPKAP kinase 2 (MAPKAP-K2) pathway alters its subcellular localization, and p38 kinase/ MAPKAP-K2 signaling is known to regulate mRNA stability and translation, as well as other biological processes. Phosphorylation of the Ago/microRNA complex and associated proteins by p38/MAPKAP-K2 may promote or prevent assembly of repressive complexes on specific mRNAs, leading to the modulation of microRNA-mediated repression on specific subsets of targets in neurons. We propose to use a recently developed method for biochemical purification and high-throughput sequencing of mRNA fragments associated with the Ago-2 protein to identify microRNA target sites at which Ago-2/microRNA binding is modulated by activation or inhibition of the p38 pathway in primary cerebellar granule neurons. We will further characterize selected target mRNAs that exhibit modulated microRNA binding. Modulation of microRNA binding and repression by signaling pathways would significantly expand the scope of biological processes that could be regulated by microRNAs, and may also explain why some predicted microRNA target sites fail to mediate repression. Both microRNAs and the p38 signaling pathway have been implicated in multiple diseases of the nervous system, and p38 plays critical roles during responses to cellular stress and/or apoptosis, physiological processes that are also linked to microRNA functions. Demonstrating that microRNA function can be rapidly modulated in the CNS by signaling, independent of microRNA expression levels, would impact both our understanding of microRNA biology in the nervous system, and of the role of microRNAs and p38 signaling in neurological disease. PUBLIC HEALTH RELEVANCE: Neurological diseases are serious human health problems that affect numerous individuals. The research in this proposal is directed at understanding genetic regulatory mechanisms in nerve cells. The proposed studies will determine whether two distinct mechanisms interact to control genes in nerve cells. This information should contribute to our knowledge of neural development, physiology, and cellular function, as well as our understanding of neurological diseases, and it should help to provide a basis for the development of rational interventions for treating or preventing neurological diseases.

DESCRIPTION (provided by applicant): Glycation is the non-enzymatic glycosylation of sugars with proteins, lipids and DNA that lead to the production of reactive metabolites called advanced glycation end products (AGE's). Glycation occurs whenever excessive sugars are available and drives many of the complications associated with diabetes. Tumors are also characterized by high glucose levels which fuels high rate glycolysis for energy production (known as the Warburg effect). Project SuGAR is a community based research database focusing on Sea Island families affected by type-2 diabetes. The goal of this study is to use this unique resource and its banked biological samples to explore if high AGE metabolite levels predict cancer incidence and mortality in a background of normal sugar levels (non-diabetic Sea Islanders) and high sugar levels (Sea Islanders with type-2 diabetes). Recent studies have led to our hypothesis that elevated levels of AGE's promote cancer disparity through the activation of the AGE-RAGE signaling axis to promote inflammation. Specific aim 1 of this study relates secretory AGE levels to cancer incidence and mortality in Project SuGAR participants with and without type-2 diabetes. This initial study represents the first analysis of cancer incidence and mortality as well as cancer/diabetes co-morbidity within the Sea Island population of South Carolina. Ages mediate many of their deleterious effects by functioning as ligands for the receptor for advanced glycation end products (RAGE). RAGE is an oncogenic transmembrane receptor which promotes inflammatory responses. Secreted RAGE (sRAGE) is a splice variant of RAGE which can act as a decoy domain receptor to decrease AGE cellular binding of RAGE. Higher sRAGE levels are associated with favorable outcome in many tumor types. Specific aim 2 will measure sRAGE as well as inflammatory marker expression with which to relate to AGE levels and cancer incidence and mortality within the Sea Island population of South Carolina. Understanding biological links between diabetes and cancer is particularly confounded by the lack of information on potential shared risk factors. By developing the existing datasets contained within Project SuGAR to include cancer incidence and mortality rates, we will develop a unique resource to not only address these confounding factors but to analyze racial specific factors promoting disparity in diabetes, cancer and comorbidity for the two diseases. Additionally, the AGE-RAGE-inflammation signaling axis may have potential impact as prognostic/diagnostic markers to guide treatment strategies for aggressive disease. It may also define a novel area of therapeutic intervention which may be developed as clinical trials within Project SuGAR and the Sea Island community. African Americans have increased risk to develop and ultimately die of both diabetes and cancer. The development of the Project SuGAR resource and a greater understanding of the role of glycation in cancer have the potential to significantly impact survival and quality of life within this population.

? DESCRIPTION (provided by applicant): AGEs and Race Specific Tumor Immune Response in Prostate Cancer African American (AA) prostate cancer patients are more likely to die of their disease than any other race or ethnic group in the US. Here in South Carolina (SC), age-adjusted prostate cancer incidence rates are 78% higher among AA men than EA men and mortality rates three times higher. While quality of care issues and socioeconomic status clearly contribute to cancer health disparities it is becoming increasing clear that molecular and genetic differences in tumor biology also play a critical role. Glycation is the non-enzymatic glycosylatio of sugars with proteins, lipids and DNA that lead to the production of reactive metabolites called advanced glycation end products (AGE's). AGEs accumulate in our tissues as we age to promote diseases associated with growing older such as diabetes and cardiovascular disease. Glycation occurs during normal metabolism but factors associated with cancer disparity such as poor diet and a lack of exercise significantly increase the accumulation of AGEs in our bodies. This study will conduct mechanistic research to investigate AGE accumulation as a biological consequence of the factors known to contribute to prostate cancer disparity. Our recent studies have led to our hypothesis that: Race specific elevations in AGEs alter tumor associated immune responses in prostate cancer. AGEs function as a ligand activator for RAGE which is expressed on the surface of most immune cells. RAGE stimulation by AGE induces the transcriptional activation of a number of factors critical for the generation of an inflammatory environment including NFkB, STAT3 and HIF1a (4-6). Such activation results in the expression of immune associated cytokines such as IL1, IL6 and TNFa which are critical for mediating crosstalk between cancer cells and the stroma. Aim 1 will use primary and immortalized race specific cell line models to define the mechanistic implications of AGEs to the immune response. Aim 2 will use mouse models fed high and low AGE diets to determine the contribution of dietary AGEs to immune response and prostate cancer growth in vivo. The concept suggesting that AGE metabolites may represent a biological consequence of cancer disparity is a novel approach to explaining the increased incidence and mortality figures observed within specific populations. Associating the mechanistic links between glycation and altered immune response has also not been examined especially within the context of a race specific background or the prostate tumor microenvironment. By identifying a molecular consequence of cancer health disparity this study may contribute to reducing the cancer incidence and mortality rates among minority populations and identify novel potential biomarkers and define a novel area of therapeutic potential.

? DESCRIPTION (provided by applicant): Blindness and impaired vision arising from retinal disease or damage are health problems with substantial human and economic costs. Recent advances in stem cell research suggest that retinal repair in humans will be feasible in the foreseeable future. However our understanding of retinal cell biology and development remains incomplete, limiting rational design of repair strategies. Cellular complexity is a key feature of the mammalian central nervous system, including the retina. The retina contains more than 50 different types of neurons, based on morphology, neurotransmitters, and other molecular markers. Cascades of transcription factors and various signaling pathways have been implicated in retinal cell type determination and the generation of cellular diversity in the retin. Recent studies have also implicated post-transcriptional regulation by microRNAs in the control of retinal cell identity. We have identified two related miRNAs that alter retinal development and promote amacrine interneuron formation, at the expense of other retinal cell types, when ectopically expressed in the developing mouse retina. We have used Argonaute PAR-CLIP to identify endogenous target mRNAs for these and other miRNAs in the neonatal mouse retina. Here we propose to determine the requirements for these miRNAs in amacrine cell formation, using CRISPR technology in retinas. We also plan to analyze a candidate target gene for its role in the regulation of retinal development, and we propose to analyze changes in mRNA expression to identify molecular and cellular pathways in the retina that are affected by these miRNAs. Finally, we propose to investigate the scope of miRNA regulation in retinal cells expressing the Ptf1a transcription factor, which is required for amacrine and horizontal cell formation. These studies will provide insight into the molecular mechanisms that control development and cell fate determination in the mammalian retina. They are expected to provide information that will contribute to new strategies to repair retinal tissue.

? DESCRIPTION (provided by applicant): AGEs and Race Specific Tumor Immune Response in Prostate Cancer African American (AA) prostate cancer patients are more likely to die of their disease than any other race or ethnic group in the US. Here in South Carolina (SC), age-adjusted prostate cancer incidence rates are 78% higher among AA men than EA men and mortality rates three times higher. While quality of care issues and socioeconomic status clearly contribute to cancer health disparities it is becoming increasing clear that molecular and genetic differences in tumor biology also play a critical role. Glycation is the non-enzymatic glycosylatio of sugars with proteins, lipids and DNA that lead to the production of reactive metabolites called advanced glycation end products (AGE's). AGEs accumulate in our tissues as we age to promote diseases associated with growing older such as diabetes and cardiovascular disease. Glycation occurs during normal metabolism but factors associated with cancer disparity such as poor diet and a lack of exercise significantly increase the accumulation of AGEs in our bodies. This study will conduct mechanistic research to investigate AGE accumulation as a biological consequence of the factors known to contribute to prostate cancer disparity. Our recent studies have led to our hypothesis that: Race specific elevations in AGEs alter tumor associated immune responses in prostate cancer. AGEs function as a ligand activator for RAGE which is expressed on the surface of most immune cells. RAGE stimulation by AGE induces the transcriptional activation of a number of factors critical for the generation of an inflammatory environment including NFkB, STAT3 and HIF1a (4-6). Such activation results in the expression of immune associated cytokines such as IL1, IL6 and TNFa which are critical for mediating crosstalk between cancer cells and the stroma. Aim 1 will use primary and immortalized race specific cell line models to define the mechanistic implications of AGEs to the immune response. Aim 2 will use mouse models fed high and low AGE diets to determine the contribution of dietary AGEs to immune response and prostate cancer growth in vivo. The concept suggesting that AGE metabolites may represent a biological consequence of cancer disparity is a novel approach to explaining the increased incidence and mortality figures observed within specific populations. Associating the mechanistic links between glycation and altered immune response has also not been examined especially within the context of a race specific background or the prostate tumor microenvironment. By identifying a molecular consequence of cancer health disparity this study may contribute to reducing the cancer incidence and mortality rates among minority populations and identify novel potential biomarkers and define a novel area of therapeutic potential.

Full Research Project 1: Survivorship Care Physical Activity Initiative to Reduce Disparities in HRQOL for Prostate Cancer Survivors (RELate Study) SUMMARY The late effects of prostate cancer (PCa) treatment can lead to unique physical and psychosocial issues that impact their long-term health-related quality of life (HRQOL). Increased physical activity (PA) is a modifiable risk factor which may be targeted to address PCa survivorship-related treatment outcomes and lifestyle behaviors. Increased PA may have greatest impact on outcomes for African-ancestry (AA) PCa survivors, who tend to have lower HRQOL and overall survival, and in whom factors such as a lack of exercise, poor diet, and rates of obesity are most prevalent. While it is well-known that exercise can improve prognosis among cancer survivors, little is known about the bio-behavioral mechanisms and pathways through which PA may improve HRQOL and increase overall survival. Advanced glycation endproducts (AGEs) are reactive metabolites produced during normal metabolism as well as the oxidation of biological macromolecules. Lifestyle factors such as a sedentary lifestyle, poor diet, and obesity increase AGE levels in the body. The investigators hypothesize that in a randomized trial of a human PA/dietary intervention with PCa survivors previously diagnosed with Stage I-III PCa (n=120), lower AGE levels will correlate with improved HRQOL, and will lead to differential changes in immune response between AA and European American (EA) trial participants. The investigators further hypothesize that in a mouse model intervention, the mechanistic implications of lifestyle- associated AGEs to tumor-associated immune response and tumor growth and/or progression will mimic those found in human models. Aim 1 is to complete a 1-year, 2-arm (intervention vs usual care) randomized PA/dietary intervention in men diagnosed with stage I-III PCa (co-led by Dr. M. Ahmed, SCSU and Dr. D. Turner, MUSC-HCC). Aim 2 is to evaluate the effect of lifestyle intervention on PA and diet, and define their relationship with AGEs, AGEs pre-cursors, and HRQOL in men diagnosed with stage I-III PCa (led by Dr. Ahmed, SCSU). Aim 3 is to use a spontaneous PCa lifestyle mouse model to define the role of AGE-RAGE signaling to immune-cell phenotypes and tumor progression (led by Dr. Turner, MUSC-HCC). The results could lead to innovative insights for pharmacologic and lifestyle adjustment, and could identify protective factors that may underlie observed differences in PCa treatment outcomes between AA and EA men. This project is synergistically related to the other research projects included in this application

ADVANCED GLYCATION END PRODUCT ANALYSIS SHARED RESOURCE PROJECT SUMMARY The mission of the Advanced Glycation Endproducts (AGE) Analysis Shared Resource is to provide a central resource to perform uniform, consistent, and reliable measurements of AGE metabolite levels in a cost- effective manner for all the U54 South Carolina Cancer Disparities Research Center (SC CADRE) Full and Pilot Research Projects. This new Shared Resource will also evaluate all of the samples from blacks with African ancestry (AAs) in the U54 SC CADRE Full and Pilot Research Projects for Sea Island/Gullah ancestry. This innovative Shared Resource will also provide expertise and guidance in all aspects of AGE biology for SC CADRE investigators and SC CADRE undergraduate and junior faculty Scholars. The AGE Analysis Shared Resource will work with the SC CADRE Community Outreach Core to implement strategies to disseminate and educate the general public regarding the associations among lack of physical activity and obesity, AGEs, and cancer disparities.

PROJECT SUMMARY/ABSTRACT The focus of this study is on early life factors and their effect on mammary development during puberty and how they relate to increased breast cancer risk. At this time we do not understand what biological changes occur during pubertal mammary development which leads to a greater risk of developing cancer in later life. Identifying the molecular mechanisms that cause aberrant pubertal mammary development may lead to defined strategies to reduce breast cancer burden in later life. As our bodies use the sugars that we consume for energy they generate waste chemicals known as metabolites. One such group of metabolites is known as advanced glycation end products or AGEs for short. Critically apart from their production as a result of the breakdown of sugar, AGE?s are also formed through the ingestion of food and by external environmental factors such as lack of exercise. Changes in this dynamic equilibrium causes protein dysfunction, protein crosslinking, decreased genetic fidelity, altered gene expression profiles and aberrant cell signaling. Our studies have identified in animal models that a diet high in AGEs significantly alters how the breast develops during puberty. The tumor microenvironment is now becoming recognized as having a major role in facilitating both mammary development and cancer progression, and that, alterations in stromal cell signaling can precede epithelial cell alterations and act as drivers of the tumorigenic process. Critically, the high AGE diet produces architecture in the breast that resembles pre-neoplastic lesions with hyper-proliferative structures and increased levels of stromal cells. We also show that AGE levels are significantly elevated in the circulation and tumor tissue of breast cancer patients and that AGE treatment alters cancer associated signaling pathways to promote breast tumor growth. This study aims to define the mechanism by which a high-AGE diet causes the dysregulation of the mammary gland during puberty (SA1) and adulthood (SA2) and will ask if the changes observed lead to a higher risk of breast tumor formation and growth (SA3). A greater mechanistic understanding of the link between AGE intake during puberty and increased breast cancer risk may define novel potential strategies for lifestyle and pharmacological intervention aimed at reducing breast cancer risk at a defined window of susceptibility.

Abstract/Project Summary Retinal disease or injury leading to impaired vision or blindness are human health problems that reduce quality of life, generating significant human and economic costs. Advances in gene therapy and stem cell biology have made retinal repair a feasible goal. Nonetheless, rational repair strategies are constrained by current knowledge of retinal biology and development. The complex cellular composition of the retina, like other parts of the mammalian central nervous system, is an essential component of the retina?s functional capabilities, yet remains incompletely understood. The mammalian retina includes more than 100 distinct types of neurons. The generation of this cellular diversity during retinal development depends in part on sequential cascades of transcriptional regulators as well as other factors. We found the miR-216b microRNA can influence retinal development including the formation of amacrine and bipolar interneurons. We identified a target gene for this microRNA, a transcriptional repressor in the forkhead family, Foxn3, that when inhibited using RNAi or CRISPR in the developing retina increases amacrine cell formation, and when overexpressed reduces amacrine cell formation. The target genes of Foxn3 during retinal development are not known. Here we propose to identify mRNAs regulated by either loss or gain of Foxn3 function in the developing retina. We will analyze changes in mRNA expression to identify the molecular pathways by which Foxn3 modulates retinal cell fate decisions. In addition, we will identify genomic sites at which the Foxn3 protein binds in the developing retina to find candidate target genes. We also propose to analyze retinal development in the absence of miR- 216b or the related miR-216a microRNA, to determine if these miRNAs are required for amacrine cell differentiation or other functions, and to assess the function of two miR-216a/b genetic variants that may be linked to retinal disease. These studies will provide new insights into the regulation of development and cell determination in the mammalian retina. They are expected to provide information that may be relevant to retinal disease and that may contribute to new strategies to repair retinal tissue.