Robert Smart

The North Carolina State University ????????????????Molecular Pathways to Pathogenesis in Toxicology??????????????? NIEHS T32 training grant is a long-standing, impactful and multidisciplinary initiative that has supported 130 pre-doctoral trainees and 18 post-doctoral researchers over the past 41 years. Graduates of this program conduct basic and applied research, teach at universities and colleges, evaluate product safety, and assist public agencies and private industries in resolving important public health and environmental problems. Our mission is to provide the next generation of toxicologists/environmental health science (EHS) researchers with the technical, operational and professional skills necessary to conduct high impact EHS research, communicate effectively to a wide variety of audiences, and work as part of multidisciplinary teams to understand how human health is impacted by environmental factors. In this competitive renewal, we will continue with the current Molecular Pathways to Pathogenesis in Toxicology training theme and our overarching systems biology framework. This approach aims to integrate all levels of biological organization from biomolecules to human populations to elucidate the fundamental mechanisms through which environmental stressors influence human health outcomes. We have added 10 new research-active faculty for a total of 28 mentors, enhanced our mentor training requirements, reorganized key research areas, and updated core curricula. We have enhanced our core professional development series in scientific rigor, reasoning, experimental design and methods, and data analysis and interpretation through formal training in science communication, training in grant writing, coding in ????????????????R???????????????, and methods for enhancing reproducibility. Participating mentors of this NIEHS T32 training grant are supported by research grants from NIEHS, other NIH Institutes, other federal agencies including DOD, EPA, NSF, state agencies and private foundations. Environmental health science research at NC State has never been stronger as evidenced by the renewal of our NIEHS EHS Core Center (P30) and the recent award of a Superfund Research Program (SRP) (P42). These centers serve to facilitate collaborations among participating mentors/trainees and provide access to cutting-edge institutional infrastructure and financial support to advance EHS research at NC State. Both centers serve as an extraordinary resource for trainees through access to core facilities, sponsored symposia, seminars, workshops and professional development opportunities. The NIEHS T32 training program is the foundation of NC State??????????????????s highly ranked graduate program in toxicology; our well-qualified mentors, strong pool of applicants, and rigorous and comprehensive EHS training opportunities will ensure the continued success of this initiative. We request continued support for six trainees.

The mission of the Center for Human Health and the Environment (CHHE) is to advance understanding of environmental impacts on human health. Through a systems biology framework integrating all levels of biological organization, CHHE aims to elucidate the fundamental mechanisms through which environmental exposures/stressors interface with biomolecules, pathways, the genome, and epigenome to influence human disease. CHHE will develop three interdisciplinary research teams that represent NC State??????????????????s distinctive strengths. CHHE will implement specific mechanisms to promote intra- and inter-team interactions and build interdisciplinary bridges to advance basic science discovery and translational research in environmental health science along the continuum from genes to population. These teams are; - The Molecular/Cellular-Based Systems and Model Organisms Team will utilize cutting edge molecular/cellular-based systems and powerful vertebrate and invertebrate model organisms to define mechanisms, pathways, GxE interactions, and individual susceptibility to environmental agents. - The Human Population Science Team will integrate expertise on environmental exposures, epidemiology, genomics and epigenomics to identify key human pathways and link exposure and disease across populations. - Bioinformatics Team will develop novel analytics and computational tools to translate Big Data generated across high-throughput and multiscale experiments into systems-level discoveries To further increase the impact and translational capacity of these teams, CHHE will develop three new facility cores that will provide instrumentation, expertise, and training to facilitate basic mechanism- to population-based research. - The Integrative Health Sciences Facility Core will expand the ability of CHHE members to translate basic science discoveries across species and provide mechanistic insights into epidemiological studies by partnering with: a) NC State??????????????????s Comparative Toxicogenomics Database (CTD); b) East Carolina University Brody School of Medicine and c) NC Dept. of Health and Human Services. - The Comparative Pathobiology Core will be located at NC State??????????????????s top-ranked College of Veterinary Medicine and its nationally recognized veterinary pathology group to facilitate assessment of the effects of environmental stressors in the many model organisms utilized by CHHE members. - The Systems Technologies Core will introduce state-of-the-art proteomics capabilities and dedicated bioinformatics support to expand the ability of CHHE members to analyze the Next Generation Sequencing data involving the genome, transcriptome and epigenome. As a land-grant university, NC State has an extensive and active Cooperative Extension Service network throughout North Carolina. CHHE will utilize this unique network to develop a highly effective, multi-directional Community Outreach and Engagement Core to disseminate findings that will contribute to addressing disparity in exposures and health outcomes and to educate communities about environmental influences on health. A strong Career Development Core for early stage scientists that is coordinated with a robust Pilot Project Program will support cutting-edge, collaborative and multidisciplinary environmental health projects to enhance the research success and impact of our membership. Through these activities and the purposeful interfacing of different disciplines CHHE will build on NC State??????????????????s unique research and community outreach strengths to become a premier transformative and synergistic EHS Core Center.

Only ~3% of human genome encodes protein. The remaining 97% of the human genome is referred to as noncoding DNA. Initially, much of the intergenic noncoding sequence was referred to as ????????????????junk DNA??????????????? as it was considered to have no function. While some intergenic sequences contain DNA elements important in gene regulation, many intergenic sequences can be transcribed into RNA. In fact, ~85% of the human genome is transcribed into RNA. RNAs that lack protein coding function are referred to as noncoding RNAs (ncRNAs) and of these the long noncoding RNAs (lncRNAs >200 nt) represent the majority. LncRNAs are one of the largest and more diverse classes of cellular transcripts with over 10,000 lncRNA transcripts reported in the human genome; in most cases their biological function is unknown. Emerging evidence indicates they play an important role in regulating gene expression and are associated with human diseases such as cancer, Alzheimer??????????????????s and heart disease. There is a critical need to determine whether associations of lncRNAs with specific disease are functionally significant and to define and characterize the function of lncRNAs using in vivo disease model systems. Given that the etiology of most chronic human diseases involves interactions with environment, it is also important to determine how environmental factors impact the expression, activity and function of lncRNAs. Nonmelanoma skin cancer (NMSC) is the most common cancer in the United States. The majority of NMSCs is caused by solar UVB radiation. p53 plays a key role in the response of skin keratinocytes to UVB-induced DNA damage by inducing cell cycle arrest and apoptosis. In skin cancer, the incidence of p53 mutations ranges from 50-90%. UVB-induced mutation of p53 allows keratinocytes upon successive UVB exposures to evade apoptosis and cell cycle arrest and these defects have a critical role in skin cancer development. LincRNA-p21 is a lncRNA and was recently discovered to be a direct transcriptional target of p53 where it serves as a mediator of p53-dependent transcriptional repression. We observed that; i) lincRNA-p21 is highly inducible by UVB in the mouse skin in vivo and in human/mouse keratinocytes, ii) UVB-induction of lincRNA-p21 is p53-dependent and iii) lincRNA-p21 has a key role in UVB-induced apoptotic cell death in keratinocytes. Our plan is to characterize the regulation and function of lincRNA-p21 in keratinocytes and skin in vivo in response to UVB and to define the role of lincRNA-p21 in UVB-induced skin cancer. The central hypotheses are i) lincRNA-p21 is induced by UVB in keratinocytes through a p53-dependent pathway to produce apoptotic cell death and ii) lincRNA-p21 functions as a tumor suppressor in NMSC whereby the loss of lincRNA-p21 expression allows mutant p53 and non-mutant p53 keratinocytes to evade UVB-induced apoptotic death leading to skin cancer. The proposed studies are significant as they represent the first characterization of a lncRNA function in an in vivo disease model with a highly relevant environmental component; moreover if the hypothesis is correct this will be the first demonstration of a lncRNA functioning as a tumor suppressor in vivo.

Despite advances in early detection and treatment strategies, the five-year survival rate for most squamous cell carcinoma (SCC) patients remains dismal (<10%). To identify new therapeutic targets, next-generation sequencing of SCCs revealed a significant number of activating mutations in the KEAP1-NRF2 pathway. NRF2??????????????????s activity, regulated by the ubiquitin ligase KEAP1, modulates a battery of cytoprotective genes against stress. Importantly, patients with cancers possessing NRF2 mutations display poorer survival and greater resistance to standard treatments. Thus, a need exists to elucidate the mechanisms by which mutant-NRF2 promotes cancer growth and survival. To discover mechanisms by which mutant-NRF2 drives SCC development, we generated a Nrf2E79Q/+ GEMM possessing one of the most common NRF2 activating mutations found in human SCC within the endogenous mouse Nrf2 locus. Using this model, we will determine the impact of mutant-NRF2 signaling on tumor development, progression and metastasis in the classic cutaneous SCC model. We hypothesize that mutant-NRF2 signaling will promote the progression of cutaneous SCC through novel downstream pathways. We will combine the expertise of 2 investigators, Dr. Robert Smart, a NC State Professor and Dr. Bernard Weissman, a UNC Professor, to perform the following aims: AIM#1: Determine the effects of mutant Nrf2E79Q expression on the development of squamous papillomas and their progression to SCCs and AIM#2: Identify transcription targets unique to mutant NRF2 in epidermis, papillomas and SCCs. Our research will help develop targeted drug therapies for human SCCs with frequent NRF2 mutations such as cutaneous, bladder, head and neck and esophagus.

This NIEHS training grant has been active for 30 years and has supported 102 predoctorates and 15 postdoctorates. These trainees have gone on to successful careers in toxicology in academia, industry, government and the private sector. This training program has been extensively revised to be in accord with the NIEHS mission and the new NIEHS T32 guidelines. The objective of this training program entitled, Molecular Pathways to Pathogenesis in Toxicology, is to provide trainees with the skills and knowledge necessary to investigate and elucidate how environmental toxicants/cellular stressors contribute to toxicity and influence disease outcomes. The broad overarching research theme is to understand how toxicants/cellular stressors perturb cellular signaling pathways and deregulate gene expression and how this contributes to toxicity and human disease outcomes. Specific environmental-associated human diseases/conditions such as asthma, lung fibrosis, reproductive/endocrine abnormalities, developmental abnormalities and cancer as well as reactive oxygen species (ROS)-related toxicity will be the focal molecular/cellular research areas. Examples of types of environmental agents that will be investigated include environmental carcinogens, pesticides, particulates, metals, endocrine disrupters and nanoparticles. There are fifteen preceptors from 3 colleges and 5 departments that are participating in this interdisciplinary training program. Importantly, this training grant is administered through the Department of Environmental and Molecular Toxicology which provides a structured cohesive environment for trainees. Predoctoral trainees acquire a solid background by completing coursework in toxicology, molecular biology, pharmacology, statistics, and pathology. Pre/postdoctoral trainees receive training in ethics, grant writing and oral communication skills. Pre/postdoctoral trainees participate in a weekly seminar program both as members of the audience and as presenters. Each semester a former trainee is invited back to the department to present a seminar and to share their career experiences with current trainees. A plan is in place with the College of Veterinary Medicine that will enhance the recruitment of veterinarians (DVMs) interested in human health to postdoctoral positions on this training grant. We are requesting support for 6 predoctoral trainees and 3 postdoctoral trainee positions.

GenX is a perflourinated compound used and generated in the production of non-stick coatings. This chemical has been detected in the Cape Fear River in North Carolina, upstream of where the Cape Fear Public Utility Authority obtains drinking water for the City of Wilmington (population ~250,000), Brunswick County and Pender County, NC. In June 2017, community concern about this chemical in drinking water resulted in multiple public meetings of citizens trying to obtain information about their potential exposure and resulting health effects associated with consumption of drinking water. As a result of community concern, the chemical plant has reportedly stopped discharging GenX into the river. However community concern still exists. This project is designed to help address community concerns about GenX exposure and health effects. Little is known about how GenX is stored in the body, the toxicity of GenX, or how long the chemical will remain in the environment. To address these questions, we plan to conduct a community-based study of Wilmington area residents who are served by public utility water. We will work with community partners of the Cape Fear Riverkeeper and the New Hanover County Department of Health to help identify a representative sample of residents, to collect biological samples, and to keep the community informed about what is known about GenX and what the study finds. We plan to recruit ~400 Wilmington area residents (100 men, 100 women, 100 boys, 100 girls) to provide blood, urine, and drinking water samples and to complete a questionnaire on their water use history. We plan to analyze blood, urine, and drinking water for GenX and related perflourinated chemicals; blood and urine samples will also be used for clinical tests (lipid profile, thyroid function, liver function, and urinalysis). All results from the study will be shared with both the community as a whole and each individual participant. We will have a community advisory panel for the study to help advise about study protocols, methods of reporting back results to participants, and provide guidance on ongoing or new community concerns about GenX. This project leverages the expertise of NC State??????????????????s Center for Human Health and the Environment to respond to an emerging community concern.

The transcription factor p53 can be activated by several environmental stressors including UVB solar radiation, to regulate the transcription of genes involved in senescence, apoptosis and cell cycle arrest. These anti-tumor activities of p53 are essential to prevent cancer. p53 is a tumor suppressor and approximately half of all human cancers have p53 alterations that results in loss of p53 transcriptional activity. Recently, we discovered that deletion of the C/EBP???????? transcription factor increased p53 protein levels and enhances p53 pro-apoptotic activity/apoptosis greater than 3-fold following UVB exposure. We have also found that UVB treatment induces a C/EBP????????-p53 complex and knockdown of C/EBP???????? in UVB-treated keratinocytes increases p53 protein stability. These results suggest C/EBP???????? is a negative regulator of p53 pro-apoptotic activity, through an unknown mechanism. C/EBP???????? has a key role in an environmentally-induced disease as C/EBP????????-/- mice are resistant to UVB-induced skin cancer, however it is unknown if the enhancement of p53 pro-apoptotic activity prevents the onset of UVB-induced skin cancer in C/EBP????????-/- mice. Through utilization of ChIP-seq and RNA-seq we will map both C/EBP???????? and p53 chromatin interactions and determine the effects of deletion of C/EBP???????? on the p53 transcriptional network. These studies will provide mechanistic insight into an uncharacterized function of C/EBP???????? in regulating p53 prop-apoptotic activity following UVB exposure and will enhance the foundation and fundability for our long-term goal to determine how C/EBP???????? regulates p53 activity, stability and protein-protein interactions and the importance of this function of C/EBP???????? in an environmentally-induced disease, UVB-induced skin cancer.

Cytokines play important roles in human development and disease. Specificity and cross-talk of cytokine signaling pathways appear to be important for fine-tuning stress responses and cell fate decision during development. Transforming growth factor b (TGF-b) is involved in cell growth, differentiation, tissue remodeling, immune response and angiogenesis. Interleukin 1 (IL-1) pathway plays a central role in the generation of inflammatory responses. We have found that both TGF-b and IL-1 activate TGF-b activated kinase 1 (TAK1) MAPKKK. Active form of TAK1 can enhance both TGF-b- and IL-1-dependent transcription. In response to IL-1 stimulation, TAK1 activates transcription factors AP-1 and NF-kB. While TGF-b stimulation does activate TAK1, the role of TAK1 in TGF-b signaling pathway is not known. Recently, we found that TAK1 associates with a transcriptional repressor SnoN, a negative regulator of TGF-b signaling. TAK1 induces degradation of SnoN. We hypothesize that TGF-b activates TAK1 to induce phosphorylation of SnoN and targets SnoN for proteasomal degradation, thereby up-regulating TGF-b signal transduction. In addition, we hypothesize that TGF-b and IL-1 activate TAK1 in distinct manner via specific scaffold/regulatory proteins to induce their unique cellular responses. Thus, the overall objectives of this proposal are; to delineate the pathway and functional role of TAK1 in TGF-b signaling and to elucidate the mechanisms through which TAK1 regulates signal pathway specificity. To accomplish these objectives and to test our hypotheses we will; i) determine the mechanism and role of TAK1-induced SnoN degradation in TGF-b signaling pathway; ii) isolate and characterize molecules associated with TAK1 and iii) generate a skin specific knockout of TAK1 to characterize the in vivo role of TAK1 in a tissue in which TGF-b play important roles. These studies will address unsolved questions regarding the mechanisms of TGF-b and IL-1 family signaling and will provide an understanding of the physiological function of TAK 1 in vivo.

This is a continuation to study the roles of C/EBP transcription factors in squamous differentiation and cancer.

Cancer is a disease that arises from genomic alterations in somatic cells and it is the accumulation of genetic alterations that drives tumorigenesis. Moreover, it is the rate at which a developing tumor cell acquires these genetic alterations that ultimately determines the onset of cancer. Human skin is routinely subjected to DNA damage induced by solar radiation and keratinocytes have developed intricate pathways to response to UVB-induced DNA damage. Recently, we provided the first genetic evidence CCAAT/enhancer binding protein ??A! (C/EBP??A!), a member of the basic leucine zipper family of transcription factors, functions as an epithelial tumor suppressor through utilization of mice with an epidermal-targeted ablation of C/EBP??A!. These mice are highly susceptible to UVB- and carcinogen-induced squamous papilloma development and these benign skin tumors display a highly accelerated rate of malignant progression to squamous cell carcinomas. Human skin squamous cell carcinomas and basal cell carcinomas as well as mouse skin squamous carcinomas display weak or ablated expression of C/EBP??A!. Together these findings suggest a tumor suppressor function of C/EBP??A! in skin cancer through maintenance of the genome. We hypothesize that reduced or ablated expression of C/EBP??A! results in an impaired DNA damage-induced G1 checkpoint, resulting in the accumulation of somatic mutations and promoting skin cancer progression. The overall objective of this proposal is to understand how the loss of C/EBP??A! contributes to an increased rate of malignant tumor progression focusing on the role of C/EBP??A! in the DNA damage-induced G1 checkpoint. Understanding how C/EBP??A! influences the rate of cancer progression and the acquisition of mutations will provide further insights to the mechanisms of carcinogen- and UVB-induced skin cancer as well as numerous cancers where C/EBP??A! expression is diminished. Recent analysis of the genomes from human cancers discovered that mutations in specific cancer associated genes can vary dramatically within a give tumor type and suggest the identification of the origin of genomic point mutations may be a more effective strategy for cancer treatment than targeting a specific cancer gene as mutations in other essential genes will be enhanced and selected by the mutator phenotype. Drugs that target point mutation genetic instability may delay the accumulation of mutations and subsequently prevent cancer onset.