Donna D. Zhang - US grants

DESCRIPTION (provided by applicant): The overall goal of our research is to understand the molecular mechanisms of toxicity/carcinogenicity of environmental pollutants and the endogenous cellular defense systems to cope with pollutants. Drinking water contaminated with arsenic, a known carcinogen, is a worldwide public health issue. Epidemiology studies have linked arsenic exposure to human cancers, including skin, liver, lung, kidney, prostate, and bladder cancer. Arsenic can also cause cellular damage through generation of reactive oxygen species (ROS) that are even involved in the initiation, promotion, and progression of tumors. Although arsenic is a well defined carcinogen, it is not mutagenic and induces malignant transformation possibly by an epigenetic or cell signaling mechanism. Eukaryotic cells have evolved several defense mechanisms to cope with stress from the environment, one of which is the antioxidant response utilized by mammalian cells to neutralize ROS and to maintain cellular redox homeostasis. This antioxidant system is mediated through the antioxidant response element (ARE) sequence present in the promoters of several antioxidant and Phase II detoxification genes including glutathione S-transferase, NAD(P)H quinone oxidoreductase, glutamylcysteine synthetase, and heme-oxygenase. The antioxidant response system is mainly controlled by the transcription factor Nrf2. Activated by compounds possessing anti-cancer properties, the ARE-Nrf2-Keap1 signaling pathway has been clearly demonstrated to have profound effects on tumorigenesis. More significantly, Nrf2 knockout mice display increased sensitivity to chemical toxicants and carcinogens and are refractory to the protective actions of chemopreventive compounds. Therefore, we hypothesize that activation of the ARE-Nrf2-Keap1 pathway acts as an endogenous protective system against arsenic-induced toxicity and carcinogenicity. The following specific aims are intended to further elucidate the mechanism of Nrf2-activation in protection from arsenic-induced toxicity/tumorigenicity. This knowledge can potentially serve the scientific and medical community in our objective to create novel chemopreventive agents with increased specificity and efficacy, which will have broad impact on human health worldwide. We propose to (1) determine the protective role of the ARE-Nrf2- Keap1 pathway in arsenic-induced toxicity and carcinogenicity, (2) define the molecular mechanisms of activation of the ARE-Nrf2-Keap1 pathway by arsenic, and (3) define the protective role of the ARE-Nrf2-Keap1 pathway in arsenic-induced toxicity and tumorigenicity using the Nrf2 knockout mouse model.

DESCRIPTION (provided by applicant): The transcription factor Nrf2 has emerged as a master regulator of a cellular protective mechanism by upregulating antioxidant response element (ARE)-bearing genes encoding antioxidant enzymes, detoxifying enzymes, xenobiotic transporters, and stress response proteins. Keap1, a substrate adaptor protein for a Cullin3 (Cul3)-based E3 ubiquitin ligase, tightly regulates the Nrf2-ARE signaling pathway. Under basal conditions, Nrf2 is constantly targeted for Keap1-mediated ubiquitination and subsequent proteasomal degradation to maintain a low constitutive level in al human organs. Upon activation of the pathway, the enzymatic activity of the Keap1-Cul3-Rbx1 E3 ligase is inhibited, resulting in stabilization of Nrf2 and activation of Nrf2 downstream genes. Since the discovery of the Nrf2-Keap1-ARE signaling pathway in 1999, Nrf2 has been viewed as a "good" transcription factor that protects us from oxidative stress-related diseases, including cancer. The chemopreventive property of Nrf2 has been well documented by the following two facts: (i) many of the well- studied chemopreventive compounds elicit their activities through activation of the Nrf2-ARE signaling pathway, and (ii) Nrf2-null mice are highly susceptible to chemical carcinogens and are no longer protected by chemopreventive compounds. Paradoxically, the "dark side" of Nrf2 has recently been revealed. For instance, somatic mutations that disrupt the Keap1-mediated negative regulation of Nrf2, resulting in a high constitutive level of Nrf2, have been identified in several types of tumors and cancer cel lines, especially non-small cell lung carcinoma (NSCLC). Furthermore, mounting evidence has emerged, indicating that Nrf2 contributes to chemoresistance, the major obstacle in cancer treatment. The discovery of the "dark side" of Nrf2 has clearly illustrated the urgent need to identify Nrf2 inhibitors and develop them into druggable compounds to enhance the efficacy of cancer treatments. We have screened a large number of natural products for their inhibition of ARE-luciferase activity using a stable cell line established in our lab, MDA-MB-231-ARE-Luc, containing an ARE (from GST-Ya)-dependent luciferase gene. Using this method, we have identified a plant extract that is able to inhibit ARE-luciferase activity. Furthermore, a pure compound, brusatol, has been isolated from the extract and has been found to inhibit the protein level of Nrf2 and exhibit potent anti-cancer activities. So far, we have obtained a substantial amount of preliminary data demonstrating that brusatol sensitizes several cancer cell lines to chemotherapeutic drugs in vitro and, more significantly, brusatol sensitizes lung cancer xenografts to cisplatin in vivo in an Nrf2-dependent manner. Based on the ability of brusatol to specifically inhibit Nrf2 and sensitize cultured cancer cells and xenografts to cisplatin treatments, we hypothesize that brusatol can enhance the efficacy of current cancer treatments by sensitizing cancer cells to chemotherapeutic drugs through inhibition of the Nrf2-dependant protective mechanism. The goal of the proposed research is to further characterize the anti-cancer properties of brusatol using a preclinical lung cancer model and delineate the molecular targets and mechanistic actions of brusatol. The proposed study will not only provide a framework for the development of this Nrf2 inhibitor into a therapeutic drug to combat chemoresistance, but also provide the first Nrf2 inhibitor for basic research in the field, both of which will have profound impacts on human health worldwide. Therefore, the following three aims will be pursued: Aim #1. Characterize the mechanistic actions of brusatol-mediated Nrf2 inhibition Nrf2 is primarily regulated by the Keap1-Cul3-Rbx1 E3 ligase at the protein level through ubiquitination and degradation. Therefore, we will investigate the effects of brusatol on the proteins that may enhance the activity of the E3 ligase, such as the protein subunits of the E3 ligase, as well as regulatory proteins that control the dynamic assembly/disassembly of the ligase complex. Aim #2. Determine the molecular targets of brusatol The target proteins of brusatol will be identified and verified. The biological functions of these proteins, in particular their crosstalk with the Nrf2 signaling pathway, wil be investigated. Most likely, these proteins will directly or indirectly regulate the Keap1-Cul3-Rbx1 E3 ligase. Aim #3. Evaluate the in vivo efficacy of brusatol using an LSL-KrasG12D/+ mouselung cancer model The feasibility of using brusatol as an adjuvant to enhance current cancer treatments and to combat both intrinsic and acquired resistance will be tested in this preclinical model that recapitulates the development and progression of human lung cancer. ! "! PUBLIC HEALTH RELEVANCE: Lung Cancer is the leading cause of cancer-related death worldwide. Little progress has been made in the treatment of lung cancer due to high resistance of lung cancer cells to chemotherapeutic treatments. Nrf2 is a protein that regulates one of the most important cellular defense mechanisms to cope with environmental toxins and chemotherapeutic drugs. Therefore, inhibition of Nrf2 represents a novel mechanism to sensitize cancer cells to chemotherapeutic drugs. This proposal focuses on the discovery and development of a phytochemical, brusatol, into a druggable compound that inhibits the Nrf2- mediated protective pathway, enhancing the effectiveness of a broad range of cancer treatments. In addition, mechanistic actions of brusatol will also be investigated.

DESCRIPTION (provided by applicant): The transcription factor, Nrf2, has emerged as the master regulator of a cellular protective mechanism by upregulating antioxidant response element (ARE)- bearing genes encoding antioxidant enzymes, detoxifying enzymes, xenobiotic transporters, and stress response proteins. Very recently, mounting evidence points to the dual function of Nrf2 in cancer. (i) In normal cells when the Nrf2- Keap1 axis is intact and basal level of Nrf2 are low, transient activation of Nrf2 by chemopreventive compounds confers protection against environmental toxins and carcinogens. (ii) In certain cancer cell lines, constitutive activation of Nrf2 creates an environment conducive for cancer cell survival. Moreover, Nrf2 contributes to chemoresistance and inhibition of the Nrf2 pathway enhances the efficacy of cancer treatments. Arsenic (As) is a human carcinogen, which causes tumors in the skin, lung and bladder. Large populations around the world are exposed to arsenic through contaminated drinking water, which imposes a major challenge to human health. However, a sufficient rodent model to study arsenic-carcinogenicity is still lacking. This competing renewal of NIH ES015010 takes advantage of a previously unrecognized role of arsenic in autophagy leading to prolonged activation of Nrf2, which was uncovered during the last funding period. We hypothesize that arsenic-mediated carcinogenicity is associated with its ability to deregulate the autophagic pathway. We believe that canonical Nrf2 inducers can alleviate this effect and thus, can be used as chemopreventive agents to counteract the damaging effects of arsenic. The following three aims are proposed: Aim 1 (in vitro): Elucidate a novel mechanism of Nrf2 induction by arsenic through deregulation of autophagy (prolonged activation of Nrf2). Aim 2 (ex vivo): Determine the role of Nrf2 in arsenic-mediated autophagosome formation and carcinogenicity using a transplantable syngeneic mouse lung cancer model. Aim 3 (in vivo): Validate the biological and pharmacological relevancy of this work. From this proposal we will (i) gain novel mechanistic insight of how arsenic deregulates autophagy, (ii) confirm the association between deregulation of autophagy and tumorigenicity of arsenic, (iii) provide new biomarkers and a sensitive animal model for arsenic carcinogenicity studies and (iv) demonstrate the potential translational impact of targeting the Nrf2 pathway using canonical Nrf2 activators to combat arsenic-induced toxicity and carcinogenicity. In addition, the syngeneic mouse lung cancer model developed will be invaluable for scientific communities studying other carcinogens. PUBLIC HEALTH RELEVANCE: Arsenic (As) is a human carcinogen, however, a sufficient rodent model to study arsenic-carcinogenicity is still lacking. This competing renewal of NIH ES015010 takes advantage of a previously unrecognized role of As in autophagy, which was uncovered during the last funding period. We hypothesize that As-mediated carcinogenicity is associated with its ability to deregulate the autophagic pathway. Consequently, we believe that canonical Nrf2 inducers can alleviate this effect and thus, can be used as chemopreventive agents to counteract the damaging effects of As. The translational value of this project is enormous based on the fact that large populations in the world are stilling drinking arsenic-contaminated water. As the saying goes, an ounce of prevention is worth a pound of cure. Furthermore, this proposed project will identify sensitive biomarkers for arsenic exposure, and develop a syngeneic mouse lung cancer model to study arsenic- carcinogenicity. In addition, this mouse model will be invaluable for scientific communities studying other carcinogens.

DESCRIPTION (provided by applicant): Colorectal cancer (CRC) is a major cause of tumor-related morbidity and mortality worldwide. Recent research strongly suggests that the redox-sensitive transcription factor Nrf2 (nuclear factor-E2-related factor 2) is a promising molecular target for chemoprevention of inflammation-driven colorectal carcinogenesis. The ground bark of Cinnamomum aromaticum (cassia) and Cinnamomum verum (Ceylon cinnamon), commonly referred to as 'cinnamon', is one of the three most consumed spices in the world, yet health effects of cinnamon consumption have remained mostly unexplored at the molecular level. Recently, we have identified the cinnamon-derived food factor cinnamaldehyde as the key principle in cinnamon powder responsible for potent induction of the Nrf2-regulated antioxidant response in human colon cells, conferring cytoprotection against subsequent oxidative and genotoxic insult. We propose exploratory research that tests the overall hypothesis that cinnamaldehyde represents a potent chemopreventive dietary factor targeting colorectal carcinogenesis through modulation of Nrf2-orchestrated cytoprotective mechanisms. To test this hypothesis, the following specific aims will be pursued: Aim #1. To define the specific molecular targets involved in Nrf2-activation by cinnamaldehyde in colorectal epithelial cells. Aim #2. To test feasibility of cinnamaldehyde-based intervention targeting colorectal carcinogenesis in an accepted murine model and to determine mechanistic involvement of Nrf2 in cinnamaldehyde chemopreventive activity. Successful completion of this project will generate critical proof-of-principle data guiding the rational design of future preclinical and clinical studies that aim at developing cinnamaldehyde for chemopreventive intervention targeting human colorectal carcinogenesis.

DESCRIPTION (provided by applicant): Arsenic and its arsenical derivatives are estimated to effect greater than 200 million people worldwide. Exposure to arsenicals comes from a number of sources such as contaminated drinking water, soil or as an airborne pollutant. Various epidemiological studies have linked chronic arsenic exposure to a number of disease states including cancer of the lungs, bladder, or skin; metabolic diseases such as diabetes; cardiovascular and other vascular diseases; and skin problems such as 'black foot disease'. In addition to the epidemiological studies there has been a great deal of effort to understand the mechanisms of pathology, but to date many questions along these lines remain obfuscated. Part of the problem with understanding arsenic toxicity is the sheer number of cellular systems that arsenic alters. For instance arsenic leads to oxidative stress, compromise of protein quality control, heat-shock response, and cell-cycle alterations to name a few. Work from our lab has identified a crucial link in the effects of arsenic on cells. Chronic treatment with low levels of arsenite (one of the oxides of arsenic) leads to a compromise of autophagy, a major protein quality control pathway. This breach comes at the step of autophagosome/lysosome fusion, leading to a build-up of autophagosomes and high levels of the autophagy specificity factor, p62. Critically, p62 contains a recognition element for Keap1, which is a substrate recognition factor in the Cul3-Keap1-Rbx1 E3 ubiquitin ligase complex. This E3 complex normally maintains a low level of the oxidative stress responsive transcription factor, Nrf2. In the presence of excess p62, Keap1 is occupied, allowing for constitutive, high level expression of Nrf2 and subsequent activation of antioxidant response element regulated genes. This high-level expression confers a growth advantage on the cells and can lead to diseases such as cancer. Despite these mechanistic leaps, it remains the mechanism by which arsenite interferes with autophagy is unknown. In the present research program we propose the hypothesis that arsenicals interfere with the AAA+ protein quality control machine, p97. This provides a critical link between arsenic and autophagy as well as other protein quality control mechanisms. To probe the detailed mechanistic underpinnings of this arsenic-mediated breach we will use an array of detailed mechanistic enzymology studies, coupled with cellular biochemistry, and in vivo studies. These efforts will be greatly aided by the multi-PI team we have assembled.

Project Summary Chronic arsenic exposure is a worldwide public health concern correlated with an increased risk of developing certain types of cancer, as well as metabolic diseases including type II diabetes. Arsenic-linked type II diabetes has recently been shown among populations in the United States, Mexico, Canada, Taiwan, and Bangladesh. We have previously reported that low, environmentally relevant doses of arsenic block autophagy at a later stage. Autophagy is a key cellular degradation pathway and autophagic dysfunction is thought to be an integral part of pathogenic changes that occur in adipocytes, ?-islet cells, hepatocytes, skeletal muscle, and kidney mesangial cells during diabetes progression. We have also shown that dysregulation of the autophagy pathway results in prolonged activation of the key antioxidant transcription factor nuclear factor erytheroid-derived-2-like 2 (Nrf2). Nrf2, which is normally bound and degraded in the cytosol by Kelch-like ECH-associated protein 1 (Keap1), is upregulated at the protein level following oxidative modification of Keap1 (Keap1-C151 dependent, canonical) or by sequestration of Keap1 into autophagosomes during autophagy dysfunction (p62-dependent, non-canonical). While controlled Nrf2 activation through the canonical mechanism is protective, prolonged non-canonical activation of Nrf2 causes cellular dysfunction and tissue damage, indicative of a ?dark side? to Nrf2. Therefore, we hypothesize that the prolonged activation of Nrf2, resulting from arsenic-mediated blockage of autophagy flux, is essential for arsenic in promoting type II diabetes. This hypothesis is supported by the following evidence: 1) chronic arsenic exposure decreases glucose uptake and insulin signaling in 3T3-L1 adipocytes, 2) Keap11 KD/Lepob/ob mice, a genetic mouse model for diabetes with persistent Nrf2 activation, display impaired adipogenesis, as well as decreased insulin sensitivity and glucose tolerance compared to Lepob/ob controls, and 3) ?-cell specific knockdown of Atg7, a key autophagy initiation protein, results in increased levels of p62 and poly-ubiquitinated proteins, accompanied by ?- cell loss and decreased insulin production. We have generated a large amount of data indicating that arsenic blocks autophagy at the autophagosome-lysosome fusion step. Three core SNARE proteins Stx17, SNAP29, and VAMP8 mediate fusion, with SNAP29 mediating the interaction between Stx17 on the outer autophagosomal membrane and VAMP8 on the lysosomal membrane. We believe that genetic ablation of any of these three fusion proteins will hinder autophagosome-lysosome fusion and result in prolonged Nrf2 activation, which will mimic the effect of arsenic in promoting type II diabetes. Therefore, we propose to: Aim 1: Determine the molecular mechanism by which arsenic blocks autophagosome- lysosome fusion, leading to prolonged Nrf2 activation. Aim 2: Determine if prolonged Nrf2 activation resulting from autophagic dysfunction induces metabolic reprogramming in muscle, kidney, pancreas, liver and fat cells. Aim 3: Determine if autophagy dysfunction and prolonged Nrf2 activation phenocopy arsenic in exacerbating insulin resistance, obesity, and diabetic nephropathy using type II diabetes models in SNAP29f/f, Nrf2-/-, and SNAP29f/f/Nrf2-/- mice. Impact: A detailed and thorough understanding of autophagy dysfunction and the prolonged activation of Nrf2 in arsenic-induced metabolic disease will prove extremely valuable in the generation of preventive and therapeutic strategies, as well as in the identification of biomarkers, for the populations at risk.

? DESCRIPTION (provided by applicant): Chronic exposure to inorganic arsenic is a worldwide public health problem that has been associated to increased risks of developing cancers of the lung, skin, and bladder. Among these malignancies, arsenic- induced lung cancer presents with the highest mortality rate. The precise carcinogenic mechanism of arsenic has not yet been fully elucidated despite many years of research and the severity of the health effects associated to its exposure. We have previously reported that environmentally relevant, low doses of arsenic block autophagy, which resulted in prolonged activation of Nrf2, the main orchestrator of the adaptive antioxidant and pro-survival response. Specifically, arsenic-induced Nrf2 activation does not occur through the canonical, reactive oxygen species (ROS)-sensing mechanism but through the autophagy-dependent, non- canonical mechanism. Autophagy is a bulk degradation pathway that degrades damaged organelles and protein aggregates. Autophagy blockage can result in accumulation of defective mitochondria and excessive ROS production, which cause DNA mutations. Nrf2 activation is well known for its cellular protection against ROS stress, which can enable arsenic-exposed cells to survive and sustain gene mutations that drive malignant transformation. Therefore, we hypothesize that prolonged activation of Nrf2 resulting from arsenic-induced autophagy blockage is essential for the arsenic-mediated malignant transformation. Our hypothesis is supported by several recent publications demonstrating that the tumorigenic effect of prolonged Nrf2 activation in autophagy-deficient mice was abolished by concurrent Nrf2 knockout. Moreover, earlier studies have found high constitutive levels of Nrf2 in many cancer cells, which favor their proliferation and chemo resistance, an effect known as the dark side of Nrf2. We have generated substantial amounts of data indicating that arsenic blocks autophagy by inhibiting the autophagosome-lysosome fusion step. Three SNAREs mediate the fusion: Stx17 on the outer membrane of the autophagosome interacts through SNAP29 with VAMP8 that resides on the lysosome membrane. We believe that genetic ablation of any of these proteins should prevent the fusion of the autophagosome with the lysosome, and the effects of this ablation should mimic the effects of arsenic- mediated p62-dependent Nrf2 up regulation. To better understand arsenic carcinogenicity, we propose: Aim 1: Elucidate the detailed molecular mechanism by which arsenic blocks autophagosome- lysosome fusion. Aim 2: Determine if autophagy dysregulation and prolonged Nrf2 activation are essential for malignant transformation. Aim 3: Test the tumorigenicity of cell lines and correlate it with prolonged Nrf2 activation. Impact: A detailed and thorough understanding of the molecular events leading to the prolonged Nrf2 activation in arsenic-induced carcinogenesis will prove extremely valuable in the generation of preventive and therapeutic strategies, as well as in the identification of biomarkers, for the populations at risk.

ABSTRACT (Project 5: Zhang, Boitano, Lantz) Chronic exposure to arsenic-containing dusts from the Iron King Mine Superfund Site, and other hardrock (metal) mining sites in arid and semi-arid climate is of public health importance worldwide. Both cancer and non-malignant lung diseases are associated with chronic arsenic exposure. While arsenic is classified as a carcinogen following either ingestion or inhalation, little data exist concerning the modes of action for development of noncancerous lung diseases from exposure to arsenic containing dusts through inhalation. Noncancerous lung diseases that have been associated with arsenic ingestion include both obstructive (chronic obstructive pulmonary disease, chronic bronchitis, emphysema, bronchiectasis) and restrictive (fibrosis) lung disease. Preliminary data from Project 4 and others suggest that inhalation of arsenic associated with particulates may be an important exposure route for lung toxicity. Collectively, data indicate that arsenic exposure compromises the barrier integrity of the airway epithelium by inducing an epithelial to mesenchymal transition (EMT). This project will examine a potential intervention to block arsenic toxicity. Nrf2 is a transcription factor that is activated by oxidative stress. Activation of the canonical Nrf2 pathway leads to expression of genes that can protect against increased oxidative stress. The Zhang team's studies (and others) indicate a protective role for Nrf2 against arsenic exposure. However, the molecular mechanisms of how Nrf2 protects airway epithelial cells, specifically, how activation of Nrf2 pathways can modulate EMT and airway epithelial barrier function, is not known. The hypothesis of Project 5 is that arsenic-containing dusts cause airway epithelial dysfunction through autophagy blockage/prolonged Nrf2 activation (non-canonical); however, intermittent induction of Nrf2 (canonical) by dietary supplementation during exposure can maintain airway epithelial barrier integrity, and thus, reduce arsenic-induced lung disease. These differential outcomes are indicative of a ?dark side? of Nrf2 that may contribute to arsenic toxicity. We will examine this hypothesis in the following three Specific Aims. In Aim 1, we will determine the protective role of Nrf2 in maintaining airway epithelial barrier integrity in response to dust particles with/without arsenic in vitro. We will then, in Aim 2, determine the molecular mechanisms of Nrf2 induction (canonical vs non-canonical) by dust particles with/without arsenic in vitro. Finally, in Aim 3, we will examine the efficacy of prophylactic canonical Nrf2 activation by dietary supplementation in maintaining airway epithelial barrier integrity and ameliorating lung damage in mice exposed to inhaled dust particles with/without arsenic. Impact: Dietary Nrf2 activation may counteract arsenic-mediated inhalation toxicity to lung epithelium, providing an intervention for populations at high risk of arsenic exposure. A detailed understanding of the mechanism of Nrf2 activation by arsenic dusts and its effects on airway epithelial cells will prove extremely valuable in the generation of preventive and therapeutic strategies for the populations at risk of exposure to arsenic, and potentially, other metal(oids).

SUMMARY (Project 1: Donna Zhang) Contamination of soil and water by metal-containing hazardous substances, particularly at sites near mine tailings and smelters, has led to chronic exposure of nearby communities to toxic metal mixtures, posing a serious health problem. Based on data from the Agency for Toxic Substances Disease Registry, the number one contaminant associated with mine tailings at these sites is the toxic metalloid arsenic (As). Epidemiological studies have demonstrated a positive correlation between chronic As exposure, either through drinking water or food, with an increased incidence of diabetes. Thus, exposure to As-containing mine tailings, which could result in inhalation or ingestion of As, may be a significant contributor to enhanced risk of disease in exposed communities. Importantly, despite the known severity of the health effects, the molecular mechanisms by which As-containing mine tailings enhance diabetic phenotypes have not yet been elucidated. Previously, we reported that low, environmentally relevant doses of arsenic block autophagy, a key cellular degradation pathway critical to maintaining proteostasis. Furthermore, we have shown that autophagic dysfunction results in prolonged activation of the key antioxidant transcription factor NRF2. Normally maintained at low levels through KEAP1-mediated ubiquitination and degradation by the 26S proteasome, NRF2 is upregulated at the protein level via oxidative modification of KEAP1 (KEAP1-C151 dependent, canonical) or sequestration of Keap1 into autophagosomes during As-induced autophagy dysfunction (p62-dependent, non-canonical). While controlled Nrf2 activation through the Keap1-C151 dependent canonical mechanism is protective, prolonged p62-dependent non-canonical activation of NRF2 during As exposure causes cellular dysfunction and tissue damage, indicative of a ?dark side? to NRF2. We hypothesize that As-containing mine tailings promote diabetes through p62-dependent, prolonged activation of Nrf2. This hypothesis is supported by our preliminary data indicating that wild type (WT) mice exposed to As showed impaired glucose tolerance and enhanced insulin resistance, which was not observed in Nrf2-/-, p62-/-, or Nrf2-/-p62-/- mice. Our recent RNAseq data generated from the liver of mice exposed to As for 20 weeks also showed significant changes in the expression of genes involved in glucose, insulin, cholesterol, and lipid metabolism. In this application, we will test our hypothesis by: 1) characterizing the time and dose-dependent diabetogenic potential of chronic exposure to As in drinking water or mine tailing As-particles (PM10) in WT mice (Aim 1); 2) determining the role of prolonged NRF2 activation in driving As-induced metabolic reprogramming in diabetes-relevant cell lines (Aim 2); and 3) in vivo confirmation of important molecular alterations induced by As and prolonged NRF2 activity in promoting diabetes (Aim 3). A mechanistic understanding of arsenic-mediated alterations that lead to diabetes will prove extremely valuable in the generation of diagnostic, preventive, and therapeutic strategies for populations exposed to As-containing mine tailings and populations at risk of arsenic exposure.

Title: NRF transcription factors in Environmental Stress and Disease Intervention PROJECT SUMMARY My broad research program includes in-depth mechanistic investigations of arsenic pathogenesis/NRF signaling and the translation of basic mechanistic knowledge to preclinical drug development. Chronic exposure to arsenic, an environmental contaminant that affects an estimated 160 million people worldwide, is a global public health concern correlated with an increased risk of developing certain types of cancer, as well as type II diabetes. However, a critical gap still exists in our knowledge concerning the precise pathologic mechanisms of arsenic, and generation of viable therapeutic approaches. Over the past decade, my research has revealed that dysregulation of the NRF2 signaling pathway is a key driver of arsenic-based pathologies. Accordingly, my overarching vision is to harness our body's defense systems?specifically the NRF2 response?to alleviate the damage or pathogenesis induced by arsenic. Transcription factor NRF2 controls the cellular stress response following exposure to environmental insults. Since the discovery of the NRF2 pathway in 1999, NRF2 has been viewed as a ?good? transcription factor that protects against oxidative stress-related diseases, including cancer, and controlled activation of NRF2 using NRF2-inducing compounds to prevent cancer initiation is well recognized. However, in 2008 my lab unveiled the ?dark side? of NRF2?uncontrolled NRF2 activation is a driver of cancer progression, metastasis, and resistance to therapy. Furthermore, recent unpublished work from my lab has indicated that prolonged upregulation of NRF2 may also contribute to the diabetogenic effects of arsenic. Therefore, specific NRF2 inhibitors will be powerful probes for dissecting the ?dark side? role of NRF2 in disease. A big challenge in the field is that there are no NRF2-specific inhibitors available despite the efforts made. Therefore, the key scientific questions that need to be addressed, and as such are the focus of this R35 proposal, include: (i) the molecular basis of diseases associated with arsenic exposure (focusing on lung cancer and type II diabetes); (ii) the effects of environmental stress on the NRF2 signaling network; (iii) the ways by which we can harness the NRF2 response to improve human health; and (iv) the distinct roles of the cap'n'collar (CNC) family members NRF1, NRF2, and NRF3. My lab will pursue answers to these questions through innovative and rigorous experimental approaches, which will allow us to fill current gaps, advance environmental health research, and ultimately improve human health.