Michael Karin - US grants

The human growth hormone gene family is composed of six genes coding for three proteins of physiological and clinical importance: growth hormone (hGH), prolactin (hPrl) and chorionic somatomammotropin (hCS). The correct and regulated expression of these genes is important for maintaining normal growth and development, starting from embryonic life through adulthood. Five of the genes are located on human chromosome 17 within a 60 Kb cluster, where the gene order is: hGH-N, hCS-A, hGH-V and hCS-B, but the sixth gene, hPrl, is located on chromosome 6. The high degree of sequence homology (less than 93%) and clustering suggest that the hGH and hCS genes evolved recently, probably by gene amplification. In spite of their similarity and close proximity, the expression of these genes is strictly tissue specific. hGH-N coding for GH is expressed in the anterior pituitary, while the other four genes, coding for various forms of CS and a rare variant of GH, are expressed in the placenta. On the other hand, the more distantly related Prl gene is also expressed in the anterior pituitary. While the complete structure and chromosomal organization of these genes are known, the mechanisms responsible for their strict tissue specific expression are not understood. We propose to study these mechanisms first by defining the various cis-acting genetic elements responsible for the remarkable differential expression of the hGH-N and hCS-A genes and second by characterizing the trans-acting regulatory factors which recognize these cis-elements. Once such factors are found, they will be purified to near homogeneity and their biological significance will be tested in vivo and in vitro by a variety of genetic and biochemical approaches. Finally, partial amino-acid sequences of the factors, which are of primary importance for conferring tissue specific expression upon these genes, will be determined. In addition, specific antisera will be raised against these factors and together, with oligonucleotide probes which match the protein sequence, will be used for screening of cDNA libraries. We also plan to develop a new approach for detecting clones coding for DNA binding proteins, by screening expression libraries with synthetic oligonucleotide probes which match the recognition sites of the various DNA binding proteins and a genetic selection procedure that will allow direct selection of the gene coding for one of these trans acting factors. Once the appropriate cDNA clones are isolated, they will be used for isolating the regulatory genes which control the expression of the GH gene family. We hope that studying the structure, function and regulation of the regulatory genes will reveal some of the molecular mechanisms which underlie tissue specific gene-expression.

The overall goal of this project is to understand the molecular mechanisms by which heavy metal ions affect gene expression in human cells. Trace metal ions like Zn and Cu are necessary for proper function, growth and development of all living organisms, yet when present in high concentration they can lead to adverse effects. On the other hand, metals like Cd and Hg have no obvious biological role and are significant environmental toxins, teratogens and possibly carcinogens. The long term effects of imbalanced Zn and Cu metabolism and exposure to Cd and Hg are likely to be mediated by the interactions of these metal ions with the gentic apparatus. To understand how these metal ions affect the human transcriptional apparatus we have been studying the structure and expression of the human metallothionein (MT) gene family, used as a model. The transcription of these genes is induced by heavy metal responsive elements via a putative metal responsive factor (MRF) that binds to the metal responsive elements (MRE's), four of which are present in the hMT-IIA promoter. We plan to isolate the MRF, determine a partial amino-acid sequence, raise specific antisera and isolate a cDNA clone encoding this factor. The complete structure of the factor will be determined by sequencing the cDNA. Site-specific mutagenesis will be used to study its mechanism of action. Similarly, we will isolate other factors that are responsible for the basal activity of the hMT-IIA promoter and for its induction by growth factors and tumor promoters. Their corresponding cDNAs will be isolated as well, and used to determine the structure of the factors. The interactions between the various factors which serve to regulate the expression of the hMT-IIA gene will be studied in a reconstituted in-vitro transcription system. In addition, we will examine the effect of heavy metal ions on the activity of regulatory proteins that use the "Zn binding finger motif" for recognition of specific DNA sequence. For these experiments two well characterized proteins, TFIIIA and the glucocorticoid receptor, will be used as a model. The effect of Cd and Hg on their specific and non-specific DNA binding activities will be determined in order to reach a better understanding of the mechanisms by which these toxic ions can interfere with the function of the transcriptional apparatus. Completion of these studies will provide us with the required biochemcial basis for understanding the effects of beneficial and toxic heavy metal ions on the human transcriptional apparatus.

One of the major challenges facing molecular geneticists, developmental biologists, and pathologists is understanding the mechanisms which control tissue specific gene expression in higher animals. During the past few years, we have seen extensive progress in this direction. A variety of novel in vivo and in vitro systems have been developed, a number of transcription factors involved in cell-type specific gene expression were identified and the genes encoding such factors were isolated. The hormonal and developmental mechanisms controlling the activity of these regulatory genes are subject to extensive investigation as well as the alteration of these basic controls in various disease states. This proposal requests funds to finance a summer FASEB conference on the topic of Regulation of Tissue Specific Gene Expression in Higher Animals. The purpose of this meeting is to congregate researchers actively engaged in studying various aspects of tissue specific gene expression. In addition to discussing the basic mechanism of gene regulation, special emphasis will be placed on discussion of both normal and aberrant gene regulation in the following organ systems: liver and pancreas, muscle, blood and the nervous system. We will also have special sessions dealing with the regulation of development and embryogenesis. The emphasis will be on gene regulation in vertebrates and especially in mammals, however, where appropriate, we have invited leading investigators, studying gene regulation in lower organisms, which are more amenable to genetic analysis. The conference will be held at Copper Mountain, Colorado from July 813, 1990. The meeting is designed to have formal slide presentations, informal poster sessions, a workshop, periods of group discussions, and plenty of opportunity for individual interaction. The conference will summarize the current understanding of tissue specific gene expression and should lead to dissemination of new information, concepts, and methodologies to clinically related fields; suggesting new strategies for further research.

One of the major challenges facing molecular geneticists, developmental biologists, and pathologists is understanding the mechanisms which control tissue specific gene expression in higher animals. During the past few years, we have seen extensive progress in this direction. A variety of novel in vivo and in vitro in cell-type specific gene expression were identified and the genes encoding such factors were isolated. The hormonal and developmental mechanisms controlling the activity of these regulatory genes are subject to extensive investigation as well as the alteration of these basic controls in various disease states. This proposal requests funds to finance a summer FASEB conference on the topic of Regulation of Tissue Specific Gene Expression in Higher Animals. The purpose of this meeting is to congregate researchers actively engaged in studying various aspects of tissue specific gene expression. In addition to discussing the basic mechanisms of gene regulation, special emphasis will be placed on discussion of both normal and aberrant gene regulation in the following organ systems; liver and pancreas, muscle, blood and the nervous system. We will also have special sessions dealing with the regulation of development and embryogenesis. The emphasis will be on gene regulation in vertebrates and especially in mammals, however, where appropriate, we have invited leading investigators, studying gene regulation in lower organisms, which are more amenable to genetic analysis. The conference will be held at Copper Mountain, Colorado from July 8-13, 1990. The meeting is designed to have formal slide presentations, informal poster sesions, a workshop, periods of group discussions, and plenty of opportunity for individual interaction. The conference will summarize the current understanding of tissue specific gene expression and should lead to dissemination of new information and new strategies for further research.

The growth hormone (GH)gene is specifically expressed in the somatotrophs of the anterior pituitary. This highly restricted expression is largely due to the action of a cell-type specific promoter, recognized by the pituitary specific transcription factor GHF-1. During the previous grant period GHF-1 was purified to homogeneity and its complete amino acid sequence was deduced after isolation of corresponding cDNA clones. A variety of experiments carried out both in vivo and in vitro indicate that the presence of GHF-1 is positively correlated with expression of GH. Furthermore, GHF-1 appears to be responsible for activation of the GH gene during fetal development. So far, it seems that the cell-type specific expression of the GH gene is controlled by the presence or absence of GHF-1. Therefore, the key to complete understanding of the cell-type specific activation of the GH gene and differentiation of somatotrophs lies within the mechanisms that regulate GHF-1 expression. To understand how GHF-1 expression is regulated we will study the mechanisms responsible for activating transcription of the GHF-1 gene in a temporal and spatial specific manner. The cis acting elements and the proteins which recognize them will be identified and characterized in detail. Genes encoding trans-acting factors controlling GHF-1 expression will be cloned and their expression pattern during mouse development will be analyzed. We will also investigate modulation of GHF-1 expression by various hormone signals. GHF-1 expression is also controlled post- transcriptionally probably at the level of translation. The mechanism and factors involved in controlling GHF-1 translation will be studied using in vivo and in vitro approaches. In addition to studying how GHF-1 expression is controlled it is also important to understand how the binding of this factor to the GH promoter results in activation of GH transcription. In this respect we will determine whether ectopic production of GHF-1 is sufficient for activating transcription of GH in cells which normally do not express this gene. We will also examine the effect of GHF-1 on the - chromatin structure of the GH and identify other proteins with which GHF-l interacts to activate GH transcription. We strongly believe that completion of studies will provide us with detailed molecular knowledge of the mechanisms responsible for the highly specific manner of GH transcription and its modulation in response to various hormonal signals and trophic factors.

DESCRIPTION: Transcription factors AP-1 and NF-kB are activated by a variety of physiological and pathological stimuli. Xenobiotics and therapeutic agents also modulate the activities of these transcription factors and thereby affect expression of their target genes. AP-1 activation is linked to the control of cell proliferation and is effected by tumor promoters, while anti-tumor promoters inhibit AP-1 activity. Therefore, agents that modulate the AP-1 activity have profound effects of cell proliferation and neoplastic transformation. NF-kB activation, on the other hand, plays a key role in induction of immunity and inflammatory responses. Therefore, xenobiotics that activate NF-kB can cause inflammatory disorders, while those that interfere with NF-kB activation are immunosuppressive. Dr. Karin proposes to examine the molecular mechanisms by which xenobiotics affect AP-1 and NF-kB activities, by studying their effects on several protein kinases, JNK, p38 and IkB kinase that play key roles in AP-1 and NF-kB activation. These studies will provide molecular explanation to effects of various xenobiotics, including alkylating agents, oxidants and antioxidants on cellular gene expression. There are conflicting indications that AP-1 and the protein kinases that stimulate its activity, JNK and p38, could be involved in either protection against adverse environmental conditions or in induction of apoptosis and cytotoxicity. He will examine the physiological role of AP-1 and proteins that regulate AP-1 activity in conferring resistance or sensitivity to various drugs by the use of cell lines derived from "knockout" mice deficient in these factors. More specifically, he will: 1) Determine whether JAB1 (a coactivator for c-Jun or JunD) and its targets are involved in conferring drug resistance in mammalian cells; 2) Determine the mechanism by which certain translation and glycosylation inhibitors lead to activation of JNK and p38; 3) Determine the mechanism by which alkylating agents lead to JNK and p38 activation; 4) Investigate the role of c-Jun and AP-1 in cellular sensitivity or resistance to alkylating agents; 5) Investigate the role of JNK and p38 in mediating cellular responses to alkylating agents and other xenobiotics and; 6) Identify the IkB kinase and investigate its regulation by oxidants and antioxidants.

A major goal of this Project is to identify biochemical and molecular parameters that correlate with the development of hypertension in spontaneously hypertensive rats (SHR) and could therefore be used to follow the segregation of hypertension causing genes in SHRxWKY F2 progeny and SHRxBN recombinant inbred strains. A second major goal of this unit is to identify molecular mechanisms involved in modulation of gene expression in response to target organ damage. The basic working hypothesis guiding this unit and examined by it is that stress activated protein kinases, such as JNK, play major roles in modulating cellular gene expression and phenotypes in response to stresses such as pressure overland and ischemia- reperfusion. These protein kinases carry out such functions through phosphorylation of various components and regulators of transcription factors AP-l and NK-kB, as well as regulatory proteins, such as the Na+/H+ exchanger. The same signaling pathways can also affect the proliferation of smooth muscle cells and fibroblasts and control the deposition of extracellular matrix. These signaling pathways linking extracellular events to the control of gene transcription and cellular homeostasis could therefore be of importance both in affecting the development of hypertension in response to external stress and in mediating target organ damage in response to hypertension and pressure overload. To follow these general goals and test its working hypothesis, this unit will pursue the following specific aims: 1) Elucidate the signal transduction pathways that lead to activation of stress responsive protein kinases and transcription factors following ischemia-reperfusion; 2) Determine which mitogen activated protein kinase (MAPK) family member is responsible for phosphorylation and stimulating the activity of the Na+/H+ exchanger; 3) Compare the activities of different MAPKs and stress regulated transcription factors in cardiac fibroblasts and myocytes derived from the WKY(LJ) and SHR(LJ) strains and use segregation analysis in collaboration with Projects 1 and 2 and Core C to determine the significance of such changes; 4) Determine the mechanisms by which thrombin, bradykinin and angiotensin II can stimulate JNK and p38 activities; 5) Generate mouse strains defective in the different MAPK signaling pathways to test their involvement in hypertension.

DESCRIPTION (Adapted from Investigator's abstract): Transcription factor NF-kB is activated by proinflammatory stimuli (IL-1, TNF, LPS, bacterial and viral infections) and, in turn, induces transcription of genes coding for mediators of immune and inflammatory responses. NF-kB is regulated by binding to the IkBs, inhibitors that retain it in the cytoplasm of non-stimulated cells. In response to proinflammatory stimuli, the IkBs are rapidly phosphorylated and degraded allowing NF-kB to translocate to the nucleus and induce gene transcription. The investigator has purified a large (900 kDa) protein kinase complex, IKK(IkB kinase), which mediated IkB phosphorylation and NF-kB activation in response to TNF and IL-1. Two of the IKK subunits were molecularly cloned. These proteins, IKKa and IKKb, are serine kinases that interact through leucine zippers and contact important regulatory subunits through helix-loop-helix (HLH) motifs. To complete the characterization of IKK and understand how it functions, he will purify and clone the remaining subunits, which will be expressed, purified and used to reconstitute the entire complex. He will study which IKK components are involved in activation and regulation of its IkB kinase activity and how they interact with IkBa. Using gene targeting techniques, he will generate mutant mice that are deficient either in expression of IKKa and IKKb or in their catalytic functions. He will use these mice to study the physiological functions of IKKa and IKKb, whether they mediate NF-kB activation in response to all known proinflammatory stimuli and whether they are responsible for phosphorylation of all IkB types. The phenotypes of these mice will also indicate whether IKKa and IKKb always function together and whether they regulate targets other than NF-kB. The investigator will also create mice with activating mutations in IKKa or IKKb, which may be prone to development of autoimmune and inflammatory disorders and thus provide useful disease models. He will study the role of protein phosphorylation in IKK activation and identify IKK kinases. He will investigate the signaling pathways leading from the IL-1 and TNF receptors to IKK by focusing on events occurring downstream to the recruitment of TRAF signal transducers.

DESCRIPTION: Exposure of mammalian cells or skin to ultraviolet (UV) radiation in DNA damage and induction of a stress response called the UV response. This induction response is mediated by several transcription factors, including AP-1, NF-kB and p53. Only p53 is likely to be directly induced in response to UV-damaged DNA. AP-1 and NF-kB, on the other hand, are activated through signal transduction cascades that appear to be elicited by effects of short wavelength UV on the cell surface, independently of DNA damage. Both pathways rely on specific signal-responsive protein kinases. The protein kinases that stimulate AP-1 activity in response to UV irradiation are JNK and p38. The protein kinase involved in NF-kB activation has not been molecularly identified. As it phosphorylates the IkB inhibitors of NF-kB, we refer to it as the IkB kinase. We propose to further investigate the mechanism by which exposure to UV leads to activation of JNK, p38 and the IkB kinase by establishing a cell-free system in which these kinases can be activated by UV. We also plan to molecularly clone the UV responsive IkB kinase and study its activation mechanism. Most importantly, however, our studies will focus on the physiological roles of these protein kinases in the response of mammalian cells to UV radiation, especially their potential involvement in protection against UV damage. This aspect of the UV response is the least well understood. Activation of AP-1 and NF-kB have both been proposed to be involved in diverse and conflicting responses including apoptosis, tumor promotion, protection against radiation induced damage and aging. As UV light is a common carcinogen and genotoxic agent to which we are exposed, understanding the function of UV activated protein kinases is of great physiological and clinical importance. Using fibroblasts and T cells derived from knockout mice that are deficient in critical components of AP-1 and NF-kB activation we will investigate whether these pathways are protective or damaging. We also plan to identify genes that are specifically induced in response to JNK and c-Jun activation by UV radiation. These studies should shed new light on the mechanisms underlying cellular responses to UV radiation. These studies will also contribute to understanding how cells respond to other forms of radiation.

DESCRIPTION: (Adapted from the Investigator's Abstract) This is a continuation application for a long-term project on effects of trace metal ions on gene expression in human cells. During the last period, we began to investigate the effects of toxic metal ions on signal transduction pathways used for global regulation of gene expression, cell proliferation and survival. The tumor promoter arsenite (As+3) was found to be a potent activator of two mitogen activated protein kinase (MAPK) cascades that stimulate the activity of transcription factor AP-1. This effect of As+3 is due to inhibition of a dual-specificity protein phosphatase, JNK phosphatase, whose normal function is to keep the MAPKs, JNK and p38 in a low activity state. As induction of AP-1 activity is closely linked to tumor promotion, the JNK phosphatase is probably an important mediator of As+3 cocarcinogenesis. It may also be involved in As+3 induced inflammatory disease. To examine the physiological role of the JNK phosphatase we will characterize and molecularly identify it using a combination of biochemical and molecular biological approaches. Dominant-negative mutants will be transfected into cultured cell lines to inhibit endogenous JNK phosphatase activity and thus assess its function in cell physiology. We will also examine the susceptibility of mouse strains and cell lines deficient in JNK or JNK phosphatase to As+3 induced cocarcinogenesis and toxicity. As+3 and other trace metals were also proposed to act through non-specific induction of oxidant stress. We therefore started to investigate the regulation of transcription factor NF-KB, which was proposed to be a major sensor of oxidant stress. We recently purified and cloned a key component in the pathway leading to NF-KB activation, the protein kinase responsible for phosphorylation and eventual degradation of the inhibitors of NF-KB, the IKBs. We now propose to study the molecular mechanism by which oxidants lead to activation of this IKB kinase (IKK). As NF-KB plays a key role in inflammation, understanding the regulation of IKK activity by oxidants, such as ozone, will provide a molecular basis for oxidant induced inflammatory disease.

DESCRIPTION: Exposure of mammalian cells or skin to ultraviolet (UV) radiation in DNA damage and induction of a stress response called the UV response. This induction response is mediated by several transcription factors, including AP-1, NF-kB and p53. Only p53 is likely to be directly induced in response to UV-damaged DNA. AP-1 and NF-kB, on the other hand, are activated through signal transduction cascades that appear to be elicited by effects of short wavelength UV on the cell surface, independently of DNA damage. Both pathways rely on specific signal-responsive protein kinases. The protein kinases that stimulate AP-1 activity in response to UV irradiation are JNK and p38. The protein kinase involved in NF-kB activation has not been molecularly identified. As it phosphorylates the IkB inhibitors of NF-kB, we refer to it as the IkB kinase. We propose to further investigate the mechanism by which exposure to UV leads to activation of JNK, p38 and the IkB kinase by establishing a cell-free system in which these kinases can be activated by UV. We also plan to molecularly clone the UV responsive IkB kinase and study its activation mechanism. Most importantly, however, our studies will focus on the physiological roles of these protein kinases in the response of mammalian cells to UV radiation, especially their potential involvement in protection against UV damage. This aspect of the UV response is the least well understood. Activation of AP-1 and NF-kB have both been proposed to be involved in diverse and conflicting responses including apoptosis, tumor promotion, protection against radiation induced damage and aging. As UV light is a common carcinogen and genotoxic agent to which we are exposed, understanding the function of UV activated protein kinases is of great physiological and clinical importance. Using fibroblasts and T cells derived from knockout mice that are deficient in critical components of AP-1 and NF-kB activation we will investigate whether these pathways are protective or damaging. We also plan to identify genes that are specifically induced in response to JNK and c-Jun activation by UV radiation. These studies should shed new light on the mechanisms underlying cellular responses to UV radiation. These studies will also contribute to understanding how cells respond to other forms of radiation.

Many Superfund site toxicants exert their chronic toxicity by causing damage to cellular macromolecules via oxidative stress. Exposure to such pro-oxidants also results in induction of a gene expression program whose primary function is to protect cells from oxidative stress. Little is known about the components of the mammalian oxidative stress response and its regulatory logic. To rectify these deficiencies, Project 1 will use a variety of genetic, cell biological and biochemical approaches to investigate the role of already identified stress activated protein kinases in the mammalian response to oxidative stress, identify new components of this induction responses and determine the mechanism of gene induction by a few model toxicants found at Superfund sites, such as arsenite and carbon tetrachloride. We will also search for new regulatory molecules, including protein kinases and transcription factors, involved in the oxidative stress response. Once identified, the pathophysiological function of these molecules will be analyzed through generation of constitutive and conditional knockout mouse mutants. We will investigate how such genetic alterations affect the ability of these animals or cells derived from them to withstand exposure to Superfund site toxicants that are believed to act via induction of oxidative stress. In addition to elucidating the basic regulatory logic underlying the mammalian response to oxidative stress, this project will have two practical outcomes relevant to the mission of the Superfund Research Program; it will create: 1) gene arrays, cell lines and transgenic mice that can be used as biosensors for monitoring exposure to toxicants that cause oxidative stress; 2) strains of mice that are deficient in activation of the protective response to oxidative stress. Such mice should be supersensitive to pro-oxidants and thus will facilitate the detection and evaluation of new suspected toxicants and mixtures of chemicals from Superfund Sites for their ability to cause oxidative stress mediated toxicity. To accomplish these goals this project will collaborate and interact with Projects 2, 3, 4 and 5 and will rely on all research support cores.

DESCRIPTION (provided by applicant): Bacillus anthracis, the causative agent of inhalation, cutaneous, and gastrointestinal anthrax, is an aggressive pathogen, whose unique properties make it an ideal bioterrorism agent. After inhalation, B. anthracis spores germinate in alveolar macrophages, which carry the bacteria to lymph nodes where they replicate and eventually disseminate through the blood stream. During this process, B. anthracis kills the macrophage and thereby evades detection and attack by the host innate immune system. Macrophage killing is mediated by the lethal toxin (LeTx), whose active subunit is lethal factor (LF). Curiously, at very low concentrations, LF activates the macrophage to produce proinflammatory mediators, whereas at higher concentrations it displays selective cytotoxicity toward macrophages. We found that modest concentrations of LF selectively induce the apoptosis of activated but not resting macrophages. This process is likely to depend on the ability of LF to cleave MAPK kinases (MKKs ) at a site required for MAPK activation. Our results suggest that activation of p38 MAPKs together with the activation of NF-(B is necessary for prevention of activation-induced death of macrophages. We plan to test the hypothesis that LF specifically kills activated macrophages by targeting this anti-apoptotic mechanism. We also plan to better understand why at low concentrations LF activates, rather than kills, macrophages. We therefore plan to pursue the following specific aims: 1) Determine the signal transduction mechanism by which very low concentrations of LF induce macrophage activation and production of proinflammatory cytokines; 2) Determine the mechanisms by which modest concentrations of LF induce the apoptosis of activated macrophages; 3) Identify which NF-(B target genes protect activated macrophages against LF-induced apoptosis and which genes are induced in response to very low, non-cytotoxic, concentrations of LF to mediate inflammatory shock; and 4) Identify the mechanism for activation-induced apoptosis of macrophages that lack NF-(B or p38 activity. In addition to providing a much better molecular understanding of the interaction between B. anthracis and its host, this project should enable the design of new therapeutic strategies that will tilt the balance in favor of the host macrophage in the battle against B. anthracis and inhalation anthrax.

DESCRIPTION (provided by applicant): This is a revised application for renewal of an ongoing project whose goal is to determine the mechanisms and physiological functions of radiation activated stress responses in mammals. We will continue to study signaling and gene expression responses that are activated by short wavelength ultraviolet (UV) radiation, a most common environmental carcinogen. The proposed research will focus on the regulation and function of the JNK and p38 MAP kinase (MAPK) cascades that are strongly activated by UV exposure. Both of these cascades lead to induction of c-Jun, a component of transcription factor AP-1 We found that c-Jun induction, a hallmark of the mammalian UV response, is required for allowing UV irradiated mouse fibroblasts to return to the cell cycle. This function is accomplished via interference with p53-mediated transcriptional activation through an unknown mechanism. During the next project period we plan to examine whether c-Jun and JNK exert a similar function in another cell type that is a target for UV induced carcinogenesis - the epidermal keratinocyte - and will continue to study the mechanism by which c-Jun suppresses p53 transcriptional activity. We will also investigate the role of p38 MAPK activation in modulating the outcome of UV exposure in mouse fibroblasts and will compare the mechanisms by which p38 affects cell cycle progression in UV irradiated cells to those that are used by JNK and c-Jun. In addition we will continue to elucidate the mechanisms responsible for activation of SNK and the closely related p38 MAPKs in response to UV radiation. The latter will be examined through a functional genomic approach based on the use of interfering RNA (RNAi) to systematically ablate the expression of genes coding for MAPK kinase kinases (MAPKKKs). This will allow us to identify the MAPKKK that mediates JNK and p38 activation by UV. The in vivo analysis of JNK and c-Jun function will be addressed through a reversed genetic approach based on conditional gene targeting in mice, that will allow us to abolish the expression of these proteins in a cell type specific manner without compromising animal viability.

DESCRIPTION (provided by applicant): Genetic susceptibility factors determine the propensity of individuals and populations to serious chronic diseases such as type 2 diabetes, asthma, inflammatory bowel disease (IBD), rheumatoid arthritis (RA) and atherosclerosis. It is also clear that environmental factors are probably as important as the patient's genetic makeup in determining the risk, development and progression of these major, currently incurable diseases. However, it is not yet understood how environmental and genetic factors communicate and cooperate in the pathogenic process. We propose that stress activated protein kinases, including JNK and ZKK, provide a crucial link between the environment and the genome and are important contributors to the etiology of major degenerative, inflammatory and metabolic diseases. Although this hypothesis is supported by circumstantial and preliminary evidence it requires critical and rigorous examination. We propose to use mutant mice (both constitutive and conditional knockouts, as well as knockin mutants) lacking JNK1, JNK2 or IKK-beta (the crucial catalytic subunit of the IKK complex) to evaluate the role of these protein kinases in development of obesity-induced insulin resistance and type 2 diabetes, asthma and chronic airway remodeling, and IBD. Both genetic and environmentally induced mouse models for these diseases will be used. In addition to examining the role of JNK and IKK in disease etiology and pathogenesis via loss-of-function mutations we will examine whether the constitutive activation of IKK, achieved via a novel gain-of-function approach, is sufficient to trigger the pathogenic mechanism or increase sensitivity to environmental factors. In the case of type 2 diabetes the biochemical mechanisms through which JNK and IKK contribute to disease development will be investigated in detail. Collectively, these studies will provide a molecular framework for understanding how environmental factors communicate with genetic determinants to elicit major degenerative, metabolic and chronic inflammatory diseases.

DESCRIPTION (provided by applicant): This is an application for continuation of a project whose major goal is to understand the regulation and function of the IkappaB kinase (IKK) complex and its subunits. Progress during the present funding period has been considerable - the different subunits of the IKK complex were molecularly identified and biochemically characterized, the genes encoding the different subunits were disrupted in mice and many of their physiological functions were identified. However, this progress has also generated important new questions that will be addressed during the next funding period. Most of the proposed work will focus on biochemical and functional characterization of the novel IKKalpha-dependent NF-kappaB signaling pathway that functions through regulation of NF-kappaB 2/p100 processing, identified during the current period. We will study the function of this pathway in both B lymphocytes and in the splenic stroma. In addition to studying the biochemical basis for regulation of IKKalpha-dependent pl00 processing, we will identify the genes and the transcriptional factors that are targeted by this pathway and its role in autoimmune and lymphoproliferative diseases caused by overproduction of BAFF (B cell activating factor), a member of the TNF cytokine family. We will also continue to study the pathophysiological function of the canonical NF-kappaB signaling pathway, whose activation depends on the IKKa subunit of the IKK complex. In this regard we will study the role of IKKa in intestinal epithelial cells and macrophages in innate immunity, inflammation and cancer. We will also study the mechanisms (target genes) through which IKKbeta exerts these functions. Finally, we will begin to study IKK-independent mechanisms for NF-kappaB regulation. More specifically, we will study the role of the IKK-like NF-kappaB activating kinase (NAK) in control NF-kappaB -dependent gene transcription, which may be exerted through phosphorylation of RelA and possibly other NF-kappaB proteins. As before, our approach will incorporate biochemical studies, cell biology, the construction of genetically altered mouse strains and the use of specific disease models. In addition to basic information on the regulation of NF-?B signaling, these studies will generate much needed information regarding the pathophysiological functions of these pathways relevant to several major disease areas, including cancer, autoimmunity and inflammation.

bioimaging /biomedical imaging; technology /technique development

DESCRIPTION (provided by applicant): Pathogen-induced macrophage apoptosis is an important mechanism used by several highly pathogenic bacteria to avoid detection by the innate immune system through killing of host macrophages, allowing them to establish highly virulent infections. We found that Bacillus anthracis, the causative agent of anthrax, can induce macrophage apoptosis and proposed that this process is an important contributor to its mechanism of pathogenecity. Using B. anthracis as a model, we found that induction of macrophage apoptosis requires inhibition of anti-apoptotic gene expression and activation of Toll-like receptor 4 (TLR4). Normally, TLR4 engagement results in macrophage activation and cytokine production, as well as induction of genes whose products prevent macrophage apoptosis, but B. anthracis uses its lethal toxin (LT) to inhibit activation of p38 MAP kinase (MAPK) and thereby prevents induction of macrophage survival genes. We identified several candidate macrophage survival genes whose induction is p38-dependent and is therefore LT-sensitive. We will continue with characterization of these genes and determination of their physiological role in the maintenance of macrophage survival. We also plan to identify the exact mechanism through which activation of p38 MAPK contributes to induction of these genes in response to TLR4 engagement. We have also identified the major mechanism through which TLR4 engagement can trigger macrophage apoptosis. The critical component of this mechanism is the double stranded (ds) RNA-responsive protein kinase PKR. Importantly, disruption of the gene encoding PKR protects macrophages from the apoptotic effect of B. anthracis, gersina and Salmonella. As these results were obtained ex vivo, we plan to study in detail the role of PKR in pathogen-induced macrophage apoptosis in vivo and determine whether inhibition of PKR can prevent macrophage apoptosis and reduce the burden and lethality of infections with macrophage-killing bacteria, such as B. anthracis. Assuming that PKR inhibition may provide a viable strategy for preventing pathogen-induced macrophage apoptosis, we will study in detail the mechanism through which TLR4 engagement leads to PKR activation. Interference with this activation process may be used to increase host resistance to bacterial infections, both with agents of bioterrorism and more common pathogens.

DESCRIPTION (provided by applicant): During the previous funding period the investigators have generated mice that lack the DCKP subunits, of the IKB kinase (IKK) complex only in hepatocytes. As a result these mice, called Ljver-IKKp-Knockout (LIKKO) mice, are unable to mount an NF-icB activation response in their hepatocytes. Challenge of LIKKO mice with certain hepatotoxins results in fulminant liver failure associated with a massive increase in the production of reactive oxygen species (ROS). When challenged with the chemical carcinogen diethyl nitrosamine (DEN), LIKKO mice develop 3 times as many hepatocellular carcinomas (HCC) as normal mice. The investigators proposed that LIKKO mice provide an outstanding and highly sensitive system for examining the in vivo consequences of oxidative stress caused by Superfund chemicals and for assessing their ability to cause liver cancer and/or act as tumor promoters. In addition to assessing the effect of the LIKKO mutation on the carcinogenic and tumor promoting activity of six different Superfund chemicals:arsenite, benzene, carbon tetrachloride, hexabromobiphenyl, dimethyl nitrosomine (which is closely related to DEN) and benzo(a)pyrene-7, 8-dihydrodiol-9, 10-epoxide (BPDE), they will examine the mechanism responsible for the elevated susceptibility of LIKKO mice to chemical carcinogenesis. A combination of reverse genetic and biochemical analysis will be used to examine the hypothesis that increased cell injury to the liver coupled to regenerative proliferation is the major mechanism responsible for the observed increase in chemical carcinogenesis in LIKKO mice. The researchers will examine the role played by ROS in this process and also test the hypothesis that ROS-mediated inhibition of INK phosphatases leads to prolonged INK activation after challenge of LIKKO mice with pro-apoptotic stimuli, leading to a coordinate increase in both cell death and compensatory liver regeneration. They will also test the ability of anti-oxidants to prevent these changes and reduce the incidence of liver cancer. Finally, hepatocytes from LIKKO mice will be used to develop an in vitro system for testing the hepatocarcinogenic activity of Superfund toxicants, that if successful will greatly reduce the time required for assessing the carcinogenic potential of various chemicals.

DESCRIPTION (provided by applicant): We propose that pattern recognition receptors, whose normal function is in innate immune responses, are also activated by stress-generated ligands, such as molecules released by dying cells, normal and oxidized lipids and a variety of so called "danger" signals in addition to environmental toxins and pathogen associated molecular patterns. Activation of such receptors either by endogenous- (i.e. host-generated) or pathogen- generated ligands turns on stress-activated protein kinases, such as JNK, p38 MAPK and IKK, that serve as molecular transducers that contribute to development of chronic inflammatory diseases. Importantly, these pathogenic mechanisms allow the integration of environmental factors and genetic susceptibility loci that together contribute to the development of some of the most common chronic diseases, including type 2 diabetes, asthma, inflammatory bowel disease and chronic liver disease. In the previous grant period we have generated strong evidence in support of this hypothesis by focusing on the pathogenic functions of the JNK and IKK signaling pathways. We also generated a mouse model expressing the equivalent of the most common susceptibility allele for Crohn's disease, an inflammatory bowl disease whose pathogenesis is affected by genetic and environmental factors. In the present period we will focus our main effort on the pathogenic function of different classes of pattern recognition receptors as targets for stress - and injury- generated stimuli, as well as continue with our studies on the role of p38 MAPK in liver inflammation and toxicity. More specifically we will examine: 1) the role of protein kinase C isozymes and Toll like receptors (TLRs) in obesity-induced JNK activation and insulin resistance;2) the role of TLRs in toxin-induced liver injury, liver inflammation and liver cancer;3) examine the role of p38 MAPK in toxin-induced liver injury, liver inflammation and liver cancer;4) examine the mechanism by which the intracellular NOD-like receptor NOD2 leads to activation of caspase 1 and IL-lbeta secretion;5) construct a conditional mouse mutant that allows constitutive NOD2 activation and use it along with our previously generated Nod2delta33 knockin mutant to examine effects of tobacco smoke, microparticles and bacterial products on development of NOD2- modulated colonic and airway inflammation. To accomplish these aims we will use a combination of cellular biochemistry, molecular genetics and experimental pathology, an approach that has been proven effective during the previous project period.

[unreadable] DESCRIPTION (provided by applicant): This collaborative project will investigate the role of necrosis, hypoxia and inflammation in cancer development, focusing on individual and interdependent contributions to tumor promotion and progression. We will test two related hypotheses that are based on preliminary results obtained in a model of chemically- induced liver cancer. 1. Inhibition of NF-kappaB activity in epithelial cells or carcinomas, brought about by certain cytokines or persistent NO production, can dismantle antioxidant defenses and lead to accumulation of reactive oxygen species (ROS). The latter can induce necrotic cell death through prolonged Jun kinase (JNK) activation. The necrotic death of premalignant hepatocytes during early tumor promotion and necrosis of nutrient- and oxygen-starved cancer cells during tumor progression trigger a localized inflammatory response that further augments tumor promotion and progression. 2. As solid tumors grow beyond a critical size they exhaust available supplies of oxygen, energy, nutrients and growth factors. Decreased oxygen supply results in tumor hypoxia and activation of the HIF-1 transcription factor in both cancer cells and tumor- and tissue-associated macrophages. Necrosis, causing the release of normal cellular constituents, leads to activation of tumor-infiltrating macrophages and triggers an inflammatory response dependent on transcription factor NF-kappaB. Within inflammatory cells, HIF-1 and NF-kappaB collaborate to induce expression of growth and angiogenesis factors that increase tumor blood supply and stimulate tumor growth and progression. We propose that this constant cycle of tumor growth, tumor starvation, hypoxia and necrosis, tumor-induced activation of inflammatory cells and re-stimulation of tumor growth and angiogenesis is a major and critical contributor to cancer progression. We will test these hypotheses in a mouse model of hepatocellular carcinoma (HCC) that allows critical evaluation of effects on tumor promotion, progression, and angiogenesis. Mouse molecular genetics will be used to dissect the different mechanisms involved in tumor-driven inflammation and ROS accumulation and determine their contribution to early tumor promotion as well as to tumor progression and angiogenesis. While providing new information on the role of inflammation in carcinogenesis and tumor progression, this work can lead to development of chemopreventive and therapeutic strategies based on inhibition of necrosis-induced inflammation. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Despite impressive advances in understanding cancer etiology and the development of novel therapeutic approaches, the most effective therapies for solid malignancies (carcinomas) remain surgery and radiotherapy. Although effective in removing and destroying primary tumors, most patients undergoing surgery and radiotherapy will eventually succumb to metastases that develop from residual primary cancer cells that migrate and proliferate at secondary sites. In other words, most cancer patients are killed by metastases rather than the primary tumor. Given the magnitude of this problem, research into the mechanisms that control the metastatic spread of tumors is of utmost importance and urgency This proposal is based on our preliminary results that a mutation that prevents activation of the inflammation responsive protein kinase IkappaB kinase alpha (IKKalpha) results in a dramatic decrease in the metastatic spread of prostate cancer in TRAMP mice, which express a SV40 T antigen oncogene in the prostate epithelium. This reduction in metastatic activity is associated with massive upregulation of maspin, a suppressor of metastasis whose expression is almost completely abolished in primary prostate carcinomas that express the wild type form of IKKalpha. Our preliminary results also suggest that IKKalpha activation results in inhibition of maspin expression. Given its known responsiveness to cytokines produced by stromal cells as well as by inflammatory cells, IKKalpha can provide a major conduit through which the tumor microenvironment controls the metastatic potential of prostate carcinoma cells. Our goal is to test this hypothesis and identify the molecular mechanisms through which IKKalpha controls the metastatic spread of prostate cancer and the expression of maspin in TRAMP mice. We will also examine how the tumor microenvironment modulates the activity of the IKKalpha-maspin axis and will determine the mechanisms through which maspin exerts its anti-metastatic activity in this model of prostate cancer. We will examine whether IKKalpha regulates maspin expression in human prostate cancer specimens and cell lines to control their metastatic and invasive activities. In addition to providing new and important information on the control of tumor metastasis, this work can lay the foundation to the development of novel anti-metastatic therapy based on inhibition of IKKalpha that may be applicable not only to prostate cancer but other cancers as well.

Apoptosis; Apoptosis Pathway; Autophagocytosis; CRISP; Cancers; Cell Communication and Signaling; Cell Death; Cell Death, Programmed; Cell Signaling; Cells; Chemopreventive; Chemopreventive Agent; Complex; Computer Retrieval of Information on Scientific Projects Database; Condition; Development; Eating; Electron Microscopy; Epidemiology / Surveillance; Food Intake; Funding; Grant; INFLM; Immune response; Inflammation; Institution; Intracellular Communication and Signaling; Investigators; Killings; Link; Malignant Cell; Malignant Neoplasms; Malignant Tumor; Mediating; Medical Surveillance; Mitochondria; NIH; National Institutes of Health; National Institutes of Health (U.S.); Nuclear; Pathway interactions; Research; Research Personnel; Research Resources; Researchers; Resources; Role; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Source; Surveillance; Tumor Angiogenesis; United States National Institutes of Health; anticarcinogenic; autophagy; base; biological adaptation to stress; biological signal transduction; cancer cell; host response; immunoresponse; malignancy; mitochondrial; necrocytosis; neoplasm/cancer; neoplastic; pathway; reaction; crisis; social role; stress response; stress; reaction; transcription factor; tumor

DESCRIPTION (provided by applicant): Tobacco smoking increases the risk of chronic pulmonary inflammation and lung cancer development. Much effort has been placed on identification of chemical carcinogens in tobacco smoke and monitoring their presence in smokers. However, not all smokers who are exposed to equivalent amounts of such carcinogens develop cancer. There is also a great variation in latency of lung cancer between individuals that do develop the disease. This suggests the existence of additional factors that contribute to the etiology of lung cancer. While genetic factors are likely to be of importance, based on our recent work showing a critical role for inflammation in other types of chemically-induced cancers, we propose that pulmonary inflammation is an important contributor to the pathogenesis of lung cancer. Pulmonary inflammation may also accelerate the metastatic spread of lung cancer, thereby making more than one contribution to the lethal nature of this disease. We propose that activation of the anti-apoptotic transcription factor NF-?B provides a link between chronic pulmonary inflammation caused by repetitive exposure to tobacco smoke and development and progression of lung cancer. We will use a conditional disruption of the gene encoding IKK[unreadable], a catalytic subunit of the I?B kinase (IKK) complex required for NF-?B activation, to determine the role of IKK[unreadable]-dependent NF-?B signaling in experimental lung carcinogenesis based on combined exposure to a chemical carcinogen and tobacco smoke. We will also use a conditional IKK[unreadable] deletion in myeloid cells to determine the contribution of NF-?B-driven inflammatory activation of cells such as alveolar macrophages to lung cancer development and progression. To circumvent the low efficiency of lung cancer induction by chemical carcinogens and its strong dependence on a particular genetic background we will also use transgenic models in which lung cancer is initiated by expression of K-ras or Raf-1 and will examine whether development and progression of these tumors is accelerated by tobacco smoke inhalation and other forms of pulmonary inflammation. In addition, we will use an experimental metastasis model based on orthotopic injections of Lewis lung carcinoma (LLC) cells to examine the contribution of inflammatory processes triggered either by exposure to tobacco smoke or by rapidly growing lung tumors to the metastatic spread of lung cancer. We will place special emphasis on the role of the innate immune system in the metastatic process and compare its activation by tobacco smoke inhalation to its activation by factors released by lung cancer cells. The proposed studies will provide critical and much needed information regarding the role of inflammation in lung carcinogenesis and tumor progression and should lay the foundation for anti-inflammatory therapy in prevention and treatment of lung cancer, the most prevalent cause of cancer-related mortality worldwide.

[unreadable] DESCRIPTION (provided by applicant): This is an application for continuation of a 10 year old project whose goal is to understand the regulation and function of different signaling responses controlled by the I?B kinase (IKK) complex and its three subunits: IKKa, IKK[unreadable], and IKK?. Progress during the previous project period has been considerable - in addition to improved understanding of IKK regulation and its role in NF-?B activation, we have elucidated the roles of the two IKK catalytic subunits (IKKa and IKK[unreadable]) in the control of innate immunity and inflammation as well as programmed cell death, cancer, chronic inflammatory disorders and autoimmunity. During the course of these studies, we made several unanticipated and rather surprising findings that we plan to pursue in greater detail during the next project period. These findings include the identification of the Hif1a gene, which encodes the hypoxia regulated HIF-1a subunit of the HIF-1 transcription factor, as an IKK and NF-?B regulated gene. These results suggest that in addition to its key role in suppression of apoptosis and activation of host defense and inflammation, NF-?B is also a critical regulator of the hypoxic response. In addition to studying the regulation of IKK? activity by hypoxia we will conduct studies that should clarify the pathophysiological importance of these findings in the control of innate immunity and tumor-elicited inflammation. Another unanticipated finding is the involvement of IKK?(NEMO), the IKK regulatory subunit, in activation of the JNK and p38 MAPK pathways in B cells stimulated via CD40 or BAFF receptor (BAFF-R). We will study the mechanism by which IKK? controls the activation of these pathways through formation of a signaling complex that includes TRAF2 and MEKK1, which are E3 ubiquitin ligases, and UBC13, an E2 ubiquitin conjugating enzyme. In addition we will continue to study the role of TRAF2 and its relative TRAF3 in controlling the activation of the IKK-dependent alternative NF-?B signaling pathway in response to engagement of CD40, RANK, BAFF-R and lymphotoxin(LT) a:[unreadable] receptor (LT[unreadable]R). We will focus on the role of TRAF2 and TRAF3 in controlling the turnover of NIK, a protein kinase responsible for IKKa activation. A third unexpected finding that will be followed up on is a role for activated nuclear IKKa in transcriptional regulation that is not related to its already known functions in regulation of the classical and alternative NF-?B signaling pathways. Although we first found this novel function of nuclear IKK in prostate cancer we will study its relevance to signaling by IKKa-activating receptors in cell types present within secondary lymphoid organs, such as the spleen, whose development and function are IKKa-dependent. As before, these studies will advance both our basic understanding of IKK signaling as well as its varied and wide-reaching pathophysiological functions. Project Narrative: This is an application for continuation of a 10 year old project focused on the regulation and function of the I?B kinase (IKK) complex, a master regulator of immunity, host defense and cell survival. The new studies will follow unexpected findings made during the previous project period that pertain to roles of IKK subunits in control of the hypoxic response during microbial infections and tumor-elicited inflammation and regulation of the alternative NF-?B signaling pathway, which is important for lymphoid organ development and function, as well as the pathogenesis of autoimmunity. Other new findings that will be studied in further detail are the role of the IKK? subunit in signaling to MAPK cascades during activation of antibody-producing B cells and novel nuclear functions for IKKa in cells of secondary lymphoid organs, which are important players in immunity and inflammatory diseases. [unreadable] [unreadable] [unreadable]

Bone-Derived Transforming Growth Factor; CRISP; Cancer of Prostate; Cancers; Carcinoma, Epidermoid; Carcinoma, Planocellular; Carcinoma, Squamous; Cell Communication and Signaling; Cell Nucleus; Cell Signaling; Cells; Computer Retrieval of Information on Scientific Projects Database; Development; Funding; Grant; Human; Human, General; I kappa B alpha-associated protein kinase; IKK 1 kinase; IKK alpha; IkappaB kinase alpha; IkappaBalpha kinase; Institution; Intracellular Communication and Signaling; Investigators; Light; Malignant Neoplasms; Malignant Tumor; Malignant Tumor of the Prostate; Malignant neoplasm of prostate; Malignant prostatic tumor; Man (Taxonomy); Man, Modern; Mediating; Metastasis; Metastasize; Metastatic Neoplasm; Metastatic Tumor; Milk Growth Factor; NIH; National Institutes of Health; National Institutes of Health (U.S.); Neoplasm Metastasis; Nuclear; Nuclear Translocation; Nucleus; Photoradiation; Platelet Transforming Growth Factor; Play; Post-Translational Modifications; Post-Translational Protein Processing; Posttranslational Modifications; Prostate CA; Prostate Cancer; Prostatic Cancer; Protein Modification; Protein Modification, Post-Translational; Protein Processing, Post-Translational; Protein Processing, Posttranslational; Protein/Amino Acid Biochemistry, Post-Translational Modification; Research; Research Personnel; Research Resources; Researchers; Resources; Role; Secondary Neoplasm; Secondary Tumor; Signal Transduction; Signal Transduction Systems; Signaling; Source; Squamous Cell Epithelioma; Squamous cell carcinoma; TGF B; TGF-beta; TGFbeta; Transforming Growth Factor beta; Tumor Cell Migration; Tumor Suppressor Proteins; United States National Institutes of Health; biological signal transduction; cancer metastasis; cancer type; keratinocyte; malignancy; neoplasm/cancer; new therapeutics; next generation therapeutics; novel therapeutics; social role; tumor; tumor suppressor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this proposal is to investigate the mechanism by which RIP1 promotes ROS production, thereby promoting TNF-induced cell death. Furthermore, the proposal research will provide new essential information regarding TNF signal transduction. As TNF is one of the major mediators of RA, these studies are of relevance and importance to the Arthritis Foundation.

DESCRIPTION (provided by applicant): Our goal is to understand the molecular mechanisms through which obesity and excessive fat consumption increase the risk of cancer development. Epidemiological studies have shown that of all cancers, obesity has the most dramatic effect on the common form of liver cancer - hepatocellular carcinoma (HCC). In addition to its important metabolic functions, the liver is an immune organ that is severely impacted by obesity, resulting in non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH), inflammatory conditions that greatly increase HCC risk. To genetically and molecularly investigate how obesity increases HCC risk, we established mouse models in which liver fat accumulation (hepatosteatosis) strongly enhances development of chemically-induced HCC; models that a recent review found to be highly relevant to the etiology and pathogenesis of human HCC. Using these models, we had demonstrated that obesity-induced liver inflammation plays a key role in HCC pathogenesis by activating the oncogenic transcription factor STAT3. However, hepatosteatosis also results in chronic activation of Target of Rapamycin (TOR) complex 1 (TORC1), thereby suppressing autophagy. We postulate that TORC1 activation and suppressed autophagy are additional mechanisms that contribute to the molecular pathogenesis of obesity-promoted HCC. To investigate this central hypothesis of the current proposal, four specific aims will be pursued: 1) Determine whether inhibition of TORC1 prevents obesity-promoted hepatocarcinogenesis; 2) Investigate the contribution of chronic TORC1 activation to DEN-induced hepatocarcinogenesis; 3) Identify effector pathways that mediate TORC1 effects on obesity-promoted hepatocarcinogenesis; and 4) Examine whether TORC1 feedback regulation by Sestrin has a role in obesity-promoted hepatocarcinogenesis. Whereas the first two aims will ask whether TORC1 activation is required and sufficient for obesity-promoted hepatocarcinogenesis, the third aim will explore the hypothesis that the most critical pathogenic function of chronic TORC1 activation is suppression of basal autophagy. The last aim will investigate the connection between tumor suppressor p53 and the regulation of TORC1 activity and autophagy, which depends on expression of Sestrins, a small family of p53-regulated proteins that activate AMPK. Studies will be performed mainly in vivo using both constitutive and inducible gene targeting as well as a novel approach for isolation and specific manipulation of pre-neoplastic HCC initiating cells. We will also develop new tools for uncoupling TORC1 from inhibition of autophagy. Collectively, the proposed experiments will shed new light on the pathogenic mechanisms that link hypernutrition and obesity to tumor development and progression via TORC1 and its ability to suppress basal autophagy. The proposed studies will also lead to new preventive and therapeutic strategies that will reduce the toll of obesity-promoted liver cancer as well as other cancers, such as pancreatic cancer, that are also strongly impacted by the obesity epidemic.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The goal of this collaboration with Guy Perkins of NCMIR is to investigate the mechanism by which the protein sestrin regulates target of rapamycin (TOR). The electron microscopy and mitochondrial microanatomy resources of the NCMIR will be used to elucidate structural phenotypes of sestrin knock-out skeletal and heart muscle in Drosophila fly and therapeutic intervention.

We will study how Superfund toxicants and mixtures thereof increase liver cancer risk. Our efforts will focus on specific progenitor cells responsible for generation of hepatocellular carcinoma (HCC), the most common form of liver cancer, in mice administered diethylnitrosamine (DEN), an hepatocarcinogen that represents the nitrosamine class of Superfund substances. DEN and its relative DMN undergo metabolic activation in the liver and give rise to HCC through induction of genetic alterations and compensatory proliferation that is triggered by hepatocyte cell death. We will characterize HCC initiating cells (HIC) isolated from livers of DEN-treated mice before tumors are detectable and use them to identify marker genes and genetic and epigenetic alterations that distinguish HIC from normal, non-tumorigenic, hepatocytes and fully transformed HCC cells. We will determine whether the same gene set is activated in HIC from mutant mice that spontaneously develop HCC and investigate whether signaling pathways, whose ablation augments HCC development, affect the appearance, growth and progression of HIC. We will also examine whether obesity, which increases HCC risk in humans and acts as a tumor promoter in mice, accelerates the appearance and growth of HIC and affects expression of HIC signature genes. Similar experiments will be conducted with Superfund substances that act as liver tumor promoters, such as carbon tetrachloride, polychlorinated biphenyls (PCBs) and the nuclear receptor CAR, which mediates their biological effects. We will examine whether such chemicals, alone or in combination with DEN, affect HIC formation and growth. These studies will result in identification of genetic, epigenetic and cellular programs responsible for induction and development of HCC. This knowledge and information will serve as the foundation for a new approach to rapid assessment of the hepatocarcinogenicity of diverse substances and mixtures found at Superfund sites. Also, we will work with the Research Translation Core and Community Engagement Core to share our findings pertinent to tumor promotion and obesity. High levels of obesity are a problem in Tribal and low income border communities (the vulnerable populations targeted by our SRP). These vulnerable communities may face greater risks to their health when obesity is coupled with exposure to Superfund toxicants, a point we can help the RTC and CEC communicate to broader audiences. other pollutants for their ability to induce HCC. In addition, the proposed studies may lead to new ways to prevent the occurrence of HCC, the third most deadly cancer worldwide.

DESCRIPTION (provided by applicant): Pancreatic cancer (PanCa) ranks eighth worldwide and fourth in the US as a cause of cancer deaths and is one of the most lethal cancers. New therapeutic and preventive approaches to PanCa should take into consideration the targeting of precursor lesions, intraepithelial neoplasia (PanIN), intraductal papillary mucinous neoplasm (IPMN) and mucinous cystic neoplasm (MCN), that precede invasive pancreatic ductal adenocarcinoma (PDAC), which is refractory to most currently available drugs. Although intrinsic genetic changes associated with formation of precursor lesions and their progression to PDAC are relatively well defined, the molecular mechanisms by which external factors increase PanCa risk are unknown. Major PanCa risk factors are old age, tobacco smoking, obesity, diabetes and chronic pancreatitis. It is also not clear if and how these risk factors accelerate progression of precursor lesions, which may be dormant for many years, to invasive PDAC. We have now developed two models based on targeted deletion of I?B kinase a (IKK¿) in pancreatic epithelial cells (PEC) that allow investigation of mechanisms through which risk factors, such as obesity and pancreatitis, affect development of PanIN lesions and their progression into fully invasive PDAC. Mice lacking only one Ikk¿ allele in PEC are phenotypically normal, but when placed on high fat diet (HFD) develop metaplastic PanIN lesions within four months. However, the homozygous deletion of Ikk¿ in PEC (Ikk¿?pan mice) results in spontaneous development of pancreatic fibrosis and pancreatitis in mice kept on low fat diet. When Ikk¿?pan mice are made to express a KrasG12D oncogene in PEC, they rapidly and frequently develop highly invasive PDAC at a time when wildtype mice with activated KrasG12D in PEC mainly exhibit PanIN lesions. Preliminary studies indicate that the earliest pathological changes in Ikk¿?pan mice are impaired autophagy, accumulation of the ubiquitin binding chaperone p62 and ER stress. IKK¿ downregulation and p62 accumulation were also observed in human pancreatitis, PanINs and PDAC. We therefore suspect that these changes may play an important role in PanCa pathogenesis. Accordingly, we will determine: 1) how IKK¿ controls autophagy and ER stress in pancreatic acinar cells; 2) the contributions of defective autophagy to the development of pancreatic fibrosis and accelerated malignant progression; 3) whether ER stress contributes to accelerated progression of PanCa in Ikk¿?pan mice; 4) the role of p62 accumulation in enhanced tumorigenesis in Ikk¿?pan mice; 5) how haploinsufficiency for IKKa results in PanIN lesion development upon HFD consumption and whether prolonged obesity causes eventual PanCa development in Ikk¿+/?pan mice in the absence of an activated Kras transgene. The completion of these studies will result in a much better understanding of the molecular etiology of PanCa and may lead to new strategies for blocking the progression of precursor lesions to invasive PDAC.

instmctions): The simultaneous constraint of highly efficient protective immunity together with the prerequisite for tolerance towards innocuous antigens Imposes a significant challenge for the intesfinal mucosal immune system. Several mechanisms operating in different types of DCs and/or T cells have been identified that control immune tolerance and protective immunity. TGF-|3 in particular is a crucial regulator capable of inducing FoxpS+Tregs, but paradoxically, in the presence of pro-inflammatory cytokines, promoting the conversion to Thi7 effector cells. This contrasting deviation puts TGF-(3 as a principal controller of pro- and anti-inflammatory immune responses. Recently we demonstrated that retinoic acid and other retinoids function as key regulators of the TGF-P-dependent immune deviation, capable of inhibiting the induction of proinflammatory Thi7 cells but promoting the TGF-P-dependent differentiation of anti-inflammatory FoxpS+lTregs. We further showed that in the absence of innate danger signals, retinoids can be absorbed and released by the DCs to the T cells they prime. The released retinoids then directly target differential gene expression within the T cell to promote anti-inflammatory and tolerant immunity. In the presence of danger signals sensed by the DCs, retinoids no longer suppress pro-inflammatory differentiation of primed T cells, suggesting that under innate stimulation conditions, retinoid-mediated suppressive effects on T cells are abolished and/or that retinoid signaling in the presence of innate stimuli can turn towards the DCs and promote their antigen presenting function and protective immune responses. The study proposed here, aims at elucidating retinoid-mediated mechanisms that operate in T cells or DCs and that control the balance between tolerance and protective immunity. The indicated collaborative approaches with Units 1,2 and 3 of this PPG, will allow us to extend this study and evaluate the potential cross talk between the retinoid pathways and other cellular mechanisms involved in immune regulation, as well as the role of retinoid-mediated control in directing immune responses towards specific pathogens, using various infecfious animal models. RELEVANCE (See instructions): The knowledge gained from this study will expand our understanding of regulation of pro- and anti¿ inflammatory immune responses, in particular involving mechanisms employed by the mucosal immune system to control local and systemic immunity. The study has also great potential for the identiflcation of new drug targets that may improve exisfing therapies or may lead to the development of novel medical intervention opportunities to prevent and/or treat intestinal inflammatory diseases.

DESCRIPTION (provided by applicant): This is a continuation of a long term project aimed at understanding the regulation and function of NF-?B signaling pathways in immunity and inflammation. Past achievements include elucidation of classical and alternative NF-?B signaling pathways and the mechanisms responsible for their activation. During the current project period we made several unexpected and rather surprising findings that are of great importance and we will pursue them during the next project period. Perhaps, the most surprising finding is the massive mucosal damage and intestinal epithelial cell (IEC) death caused by TNF administration to mice that express constitutively active IKK¿ in IEC. This finding seems contradictory to the well-established anti-apoptotic function of NF-?B in most cell types, including IEC, but preliminary studies confirm that the TNF-induced death of IEC that express constitutively active IKK¿ is distinct from the apoptotic cell death induced by TNF in NF-?B- deficient cells. We will investigate the mechanism responsible for this interesting phenomenon as it can explain how TNF causes damage of chronically inflamed tissues, while in normal, uninflammed, tissues, it actually provides a protective function. We expect these studies to advance the understanding of chronic inflammatory diseases, especially ulcerative colitis (UC) and Crohn's disease (CD). Another important function of NF-?B signaling is the production of key immunoregulatory cytokines, such as IL-23, in response to engagement of pattern recognition receptors (PRR). We found that IL-23 production is strongly elevated in tumor associated macrophages (TAM) of colorectal tumors relative to normal lamina propria macrophages. As the IL-23-IL-17 axis controls the progression of human and mouse colorectal cancers (CRC), we will study in detail the mechanisms that trigger its activation by identifying the PRRs responsible for tumoral IL-23 production and by investigating how colorectal tumors become permeable to TAM-activating microbial products. In this respect, we will investigate how loss of the APC tumor suppressor, NF-?B and STAT3 activation in pre-malignant IEC results in loss of junctional proteins and mucin expression, thereby contributing to barrier disruption. Similar mechanisms may underlie barrier deterioration in UC and CD, inflammatory bowel diseases that increase CRC risk. Our preliminary studies suggest that STAT3, whose activity is modulated by IKK¿/NF-?B signaling, may be a key factor in the control of epithelial homeostasis. Our preliminary studies also revealed an unexpected link between STAT3 and YAP, a transcriptional co-activator that controls tissue growth and regeneration. We will investigate how STAT3 leads to YAP activation and whether YAP mediates the effects of STAT3 on mucosal homeostasis. We will also investigate the potential cross-talk between NF-?B and YAP, which is activated during experimental colitis to control mucosal regeneration and tumorigenesis. The proposed studies will be based on a combination of biochemistry, cell biology, and mouse physiology, an integrated approach that was proven very effective in the past.

DESCRIPTION (provided by applicant): This application is built upon extensive progress made during the past five years in understanding the role of IKK? and various inflammatory and immune mechanisms in the control of metastatic progression in prostate cancer (PCa) and the development of castration-resistant tumors after androgen ablation. We found that accumulation of activated IKK? in nuclei of PCa cells is required both for metastatic dissemination and development of castration resistance. IKK? exerts its nuclear effects, in part, through transcriptional activation of the Bmi1 gene, which encodes a component of Polycomb repressive complex 1 (PRC1), as well as through repression of the metastasis inhibitor maspin. Nuclear accumulation of activated IKK? was also observed in human PCa and Bmi-1 is a key regulator of PCa stem cell renewal in both humans and mice. Therefore, we will use mouse models and human clinical specimens to investigate the mechanisms through which activated IKK? accumulates in nuclei of PCa cells and how it stimulates Bmi1 transcription. Interference with these mechanisms will offer new approaches to PCa therapy, that are supported by our preliminary results, which demonstrate inhibition of prostate stem cell activation upon loss of IKK? kinase activity and that the combination of IKK? inhibition and androgen receptor blockade completely prevents castration resistance. In PCa cells, IKK? is activated by inflammatory cytokines that are produced by immune cells which are recruited into the tumors during normal progression and after androgen ablation. We found that activated myofibroblasts are the critical source of the chemokines that recruit cytokine-producing immune cells into prostate tumors under both conditions. We will therefore study the mechanisms responsible for myofibroblast activation, both in response to androgen ablation and during normal tumor progression. We will also investigate the role of myofibroblasts in metastatic progression. Correspondingly, five specific aims will be pursued: 1) Define the mechanisms through which IKK? stimulates Bmi1 transcription in collaboration with E2F transcription factors; 2) Determine whether IKK? represses the SPB5 (Maspin) gene through its effect on Bmi-1 expression; 3) Investigate the mechanisms responsible for the nuclear accumulation of activated IKK? in PCa cells and find ways to inhibit IKK? nuclear accumulation; 4) Determine the role of myofibroblasts and their origin during metastatic progression of PCa; 5) Define mechanisms that are responsible for myofibroblast activation and appearance during development of castration-resistant cancer and metastatic progression. In addition to explaining basic mechanisms that account for PCa metastasis and emergence of castration resistance, the proposed research will lead to development of new therapeutic strategies for interference with these major causes of PCa-associated morbidity and mortality.

The IKK?NF-?B system is a master regulator of inflammation in pancreatitis. However, we have recently reported that mice with pancreas-specific deletion of I?B kinase ? (IKK?) spontaneously develop pancreatitis, revealing a new role for IKK? in maintaining normal pancreatic function. These mice, termed Ikk??pan, present with acinar cell vacuolation at 3 weeks of age and subsequently progress to pronounced pancreatic damage associated with endoplasmic reticulum (ER) stress, release of digestive enzymes into the circulation, and inflammation. By 3-5 months of age, Ikk??pan mice develop fibrosis and severe chronic pancreatitis. IKK? role in maintaining pancreatic homeostasis is independent of its kinase activity and is unrelated to effects on NF- ?B. Rather, IKK? is required for the completion of autophagy. As a result, IKK?-deficient acinar cells exhibit accumulation of autolysosomes containing partially digested organelles, leading to accumulation of the ubiquitin binding chaperone p62/SQSTM1. Pancreas-specific ablation of p62 attenuates pancreatitis in Ikk??pan mice and reduces expression of ER- and oxidative-stress markers. To further investigate the relationship between autophagy and pancreatitis, and gain better insight into IKK? function in the pancreas, we blocked the initiation of pancreatic autophagy by specific deletion of ATG7 in pancreatic epithelial cells. Atg7?pan mice also developed spontaneous pancreatitis with massive accumulation of p62 aggregates. Importantly, we found prominent accumulation of p62 aggregates in approximately 60% of human chronic pancreatitis specimens that were analyzed. We propose that detailed analysis of p62 function and downstream effectors of IKK? in exocrine pancreas will provide novel insights into the pathogenesis of human pancreatitis leading to new therapeutic and early intervention opportunities. Accordingly, we will pursue the following Specific Aims: 1). Determine the impact of p62 ablation on pancreatic homeostasis in experimental models of pancreatitis. 2). Investigate pancreatic pathology in Atg7?pan mice and determine whether it is attenuated by p62 ablation. 3). Determine the impact of ATG16L2 ablation on pancreatic homeostasis and whether its effects on pancreatic pathology are p62 dependent. 4). Investigate the pathogenic function of p62 and its underlying mechanisms. Project

DESCRIPTION (provided by applicant): Alcoholic liver disease (ALD) progresses from a normal liver, to alcoholic steatohepatitis, fibrosis and hepatocellular carcinoma (HCC). Despite of intensive studies, the pathogenesis of ALD is poorly understood due to the lack of animal models which mimic the stages of ALD progression. Furthermore, the role of IL-17 in ALD has not been evaluated. We have recently demonstrated that IL-17 signaling plays a critical role in development of liver fibrosis and cancer. Based on our preliminary data, IL-17 signaling also required for ALD, and mice devoid of IL-17 signaling develop less steatohepatitis and fibrosis. Here we propose to further explore the role of IL-17 in ALD, using an improved model of ALD in mice, which was developed as a result of a collaborative effort of Drs. Karin, Gao, Tsukamoto and Kisseleva. This model most closely reproduces the stages of ALD and reflects physiological ALD progression from steatohepatitis to alcohol-induced liver fibrosis and HCC in patients. Our central hypothesis is that IL-17 exacerbates progression of ALD from steatohepatitis to fibrosis and HCC. The goal of the study is to determine if the strategy of blocking IL-17 will have a therapeutic effect on ALD. The role of IL-17 signaling in ALD, and pathways of IL-17 regulation will be tested in this study using IL-17-/-, IL-17RA-/-, and several cell-specific knockout mice to determine the role of IL- 17 signaling 1) chronic-binge model that mimics early stages of steatohepatitis (AIM 1), 2) intragastric ethanol feeding model that mimics alcoholic steatohepatitis and fibrosis (AIM 2), and 3) diethylnitrosamine (DEN)+alcohol model that mimics alcoholic liver cancer (AIM 3). Specifically, these models will allow us to dissect specific IL-17 functions at different stages of ALD. Thus, for each model, 1) development of alcohol- induced liver injury will be determined by measuring alcohol-metabolizing enzymes dehydrogenase (ADH), cytochrome, P4502E1 (CYP2E1), expression of adipogenic genes (PPAR?, PPAR?, CEBP1), lipid peroxidation, ROS production. 2) The role of IL-17 in recruitment of inflammatory cells, Kupffer cells/macrophage activation will be determined. 3) The cellular sources of IL-17 will be identified. 4) The IL-17 target cells that critically mediate ALD progression will be determined, specific cell types will be isolated and ex vivo analyzed. 5) We will generate conditional IL-17RA-/- knockout mice in Kupffer cells, hepatocytes and Hepatic Stellate Cells (HSCs) to study their contribution to ALD. 6) The global cytokine expression profile will determined for each type of knockout mice. 7) The regulatory role of IL-23, IL-25 and IL-27 cytokines in IL-17 by Th17 cells (or other IL-17 producing cells) will be determined. These unique genetic studies will provide an insight into usefulness of IL-17 inhibitors as a novel approach to treat ALD in patients.

Abstract This is an application for continuation of a project whose long-term goal is the identification of the mechanisms through which obesity and hypernutrition enhance cancer risk. Obesity manifests one of its strongest tumor-promoting effects in hepatocellular carcinoma (HCC), the major primary liver cancer, whose US incidence has increased by 300% during the past 20 years. This increase is due to the obesity epidemic that is still spreading throughout our country. We therefore chose to address the important and general question regarding the mechanisms by which obesity increase cancer risk by studying the relationships between obesity and HCC development. In the previous project period, we developed new models for studying obesity-induced HCC and used them to establish the role of inflammation and ER stress in the progression of non-alcoholic steatohepatitis (NASH) to HCC. During these studies, we had uncovered the key pathogenic function of the autophagy receptor and signaling adaptor p62/SQSTM1, a protein that accumulates in NASH and several other chronic liver diseases, all of which increase HCC risk. We have validated the relevance of p62/SQSTM1 to the pathogenesis of human HCC and shown that its elevated expression in the non-tumor liver provides a strong prediction of HCC recurrence after curative ablation. Since all of this work was done in collaboration with Dr. Jorge Moscat, a pioneer in studying p62 function, we have decided to formalize our joint effort and pursue a more in-depth investigation of p62 involvement in obesity-induced HCC through the multi-investigator R01 mechanism. We propose to further investigate how p62 exerts its HCC-inducing activity by defining the tumorigenic function of various p62 structural motifs that mediate its interactions with other proteins. In particular, we will focus on the role of NRF2, an activator of the anti-oxidant response, and mTORC1, a major metabolism regulating protein kinase complex, as downstream effectors of p62-dependent liver tumorigenesis. We will also conduct unbiased metabolomic analysis to decipher the role of NRF2- and mTORC1-regulated metabolic pathways in obesity-induced HCC development. In addition, we will use state-of-the-art high throughput screens to search for small molecule inhibitors that target p62-driven NRF2 and mTORC1 activation and thereby prevent obesity-induced HCC development or inhibit the growth and progression of such tumors. In addition to greatly improving our understanding of the mechanisms through which obesity induces HCC development, this investigation may lead to new preventive and therapeutic strategies.

Project Summary/Abstract Project 1 will generate and use mouse models for studying how exposure to hepatotoxic Superfund substances causes fatty liver disease and its different manifestations, including inflammation, fibrosis and cancer. It has become increasingly clear that exposure to many industrial and environmental toxicants can cause toxicant- associated fatty liver disease (TAFLD) and its more serious form, toxicant-associated steatohepatitis (TASH), whose pathological signs are strikingly similar to those of non-alcoholic fatty liver disease (NAFLD) and non- alcoholic stateohepatits (NASH), respectively. As the pathogenic mechanisms underlying to TAFLD and TASH are poorly understood, we posit that better insight to toxicant-induced liver diseases can be gained from studying them along with NAFLD and NASH. Moreover, pre-existing NAFLD can increase the susceptibility of the liver to toxicant induced damage, further justifying the study of this metabolic condition that can progress to NASH or TASH and then to liver cirrhosis and cancer, diseases whose incidence is greater in economically disadvantaged Indian- and Mexican-American populations. During the past project period we obtained exciting and important preliminary results according to which the development and clinical progression of NASH and TASH are strongly modulated by two different populations of B lymphocytres, spleen-derived B2 B cells that promote fibrotic progression and IgA-producing plasmocytes that attenuate the onset of liver fibrosis, but favor the establishment of an immunosuppressive microenvironment permissive to the growth and progression of hepatocellular carcinoma (HCC). Correspondingly, the specific targeting of IgA+ plasmocytes results in immune rejection of established HCC. To better understand the role of these B populations in the development and progression of NASH and TASH and to generate a new therapeutic approach to the treatment of toxicant-and obesity-induced liver cancer we will: 1) Define the pathways responsible for B cell recruitment into toxicant-damaged steatotic livers; 2) Determine how TASH and NASH trigger IgA class switch recombination (CSR) in liver-infiltrating B cells; 3) Determine how IgA+ plasmocytes attenuate NASH- and TASH-associated liver fibrosis; 4) Determine the role of T cell subsets and microbiota in the development of NASH and TASH; 5) identify secreted metabolites that along with IgA can be used for non-invasive detection of liver fibrosis; 6) determine the role of IgA+ plasmocytes in NASH and TASH progression to HCC. Our ability to accomplish these goals and gain a much better understanding of the immune mechanisms that affect the pathogenesis of NASH and TASH will be greatly enhanced by our research cores and the close collaborative interactions with other research projects.

PROJECT SUMMARY This renewal application of our long-term effort to understand how IKK-dependent NF-?B signaling controls inflammation and immunity is focused on positive and negative regulation of the NLRP3 inflammasome by NF-?B and mitochondrial (mt) metabolism. Our effort will be placed on complete elucidation of a novel signaling mechanism, identified in our laboratory, through which engagement of Toll-like receptors (TLR) renders macrophages (M?) responsive to stress, damage signals and microparticles that trigger NLRP3 inflammasome activation and induce IL-1? and IL-18 production. Persistent NLRP3 inflammasome activation is involved in several neurodegenerative, metabolic and inflammatory diseases, e.g. Alzheimer?s disease, type II diabetes and osteoarthritis (OA), but its poor understanding has prevented development of novel NLRP3-specific anti- inflammatory drugs. Furthermore, previous attempts to alleviate inflammation by targeting IKK-dependent NF- ?B signaling have failed due to enhanced NLRP3 inflammasome activation. By studying how NF-?B negatively regulates the NLRP3 inflammasome, we identified a critical role for mitochondria in control of inflammasome activity. Whereas, NF-?B- and p62-dependent mitophagy terminates NLRP3 inflammasome activation in stimulated M?, TLR4 or TLR3 engagement triggers mtDNA replication via a novel, pathway based on activation of IRF-1 and induction of the nucleotide kinase CMPK2. This pathway is essential for production of oxidized (Ox) mtDNA in TLR-activated M? that were exposed to diverse NLRP3 inflammasome activators, e.g. ATP, nigericin, alum and DOTAP liposomes. Our results suggest that Ox-mtDNA is the ultimate NLRP3 ligand responsible for inflammasome assembly and activation. We will continue to study this pathway and investigate the suitability of its targeting for treatment of currently incurable inflammatory diseases, such as OA. Accordingly, we will determine whether CMPK2 knockout and knockin mice exhibit defective NLRP3 inflammasome activation and are therefore resistant to hydroxyapatite-induced joint inflammation. We will also determine how CMPK2- dependent mtDNA replication supports Ox-mtDNA production and examine whether the latter binds NLRP3 directly, map the binding site and conduct biochemical and structural studies to determine how Ox-mtDNA binding induces the association of NLRP3 with the inflammasome scaffold protein ASC. We will investigate how other mt signals and metabolites, reactive oxygen species (ROS) and itaconic acid (IA), modulate synthesis of pro-IL-1?, pro-IL-18 and other cytokines. These studies will focus on the role of the oxidant-responsive transcription factor NRF2 in cytokine gene expression and will also explore how mtROS and IA affect NF-?B activity and its crosstalk with NRF2. These studies will expand our basic understanding of the fundamental mechanisms that control inflammation and will lay the foundation for developing novel anti-inflammatory drugs that inhibit IL-1? and IL-18 production without interfering with antimicrobial immunity.

PROJECT SUMMARY This project will explore adaptive immune mechanisms that control development of hepatocellular carcinoma (HCC), a leading cause of cancer-related deaths, and dictate its responsiveness to PD-1:PD-L1 checkpoint inhibitors. Our team, including Michael Karin, Ph.D. and Shabnam Shalapour, Ph.D. at UCSD School of Medicine and Hidekazu Tsukamoto, D.V.M., Ph.D. and Anthony El-Khoueiry, M.D. at USC Keck School of Medicine, represents an ideal blend of basic researchers, translational scientists and oncologists who are interested in HCC molecular pathogenesis and treatment, especially in HCC caused by non-alcoholic (NASH) and alcoholic (ASH) steatohepatitis. Although the US incidence of HCC and its associated mortality have nearly tripled in the past generation, insufficient effort has been made toward identification and development of innovative and effective HCC therapies. However, the ideal and timely confluence of basic preclinical research and applied clinical studies carried out by our team members has the potential to critically transform HCC treatment forever. Together we found that chronic liver inflammation results in suppression of HCC-protective immunosurveillance to support rapid malignant progression. This unique immunopathogenic mechanism renders HCC responsive to drugs that disrupt the PD-1:PD-L1 checkpoint, but even the impressive response seen thus far and the selection of patients who will benefit from this therapeutic approach can be further improved. Such improvements can only be achieved by a deeper understanding of the mechanisms through which PD-1:PD-L1 inhibitors act, the factors that determine their efficacy, and the causes of treatment failure. We will achieve these goals through integrated studies of clinical specimens collected by Dr. El-Khoueiry and sophisticated, faithful and robust mouse models of non-viral HCC developed by Drs. Tsukamoto, Shalapour, and Karin. The immune mechanisms that control NASH- and ASH-driven HCC development in these models are highly similar to those that operate in human patients. Using this integrated approach, we will pursue five specific aims: 1) determine whether serum IgA concentrations correlate with therapeutic response to PD-1 blockade in patients with non-viral HCC; 2) develop reliable mouse models of ASH-driven HCC; 3) compare the immunosuppressive mechanisms that contribute to development of NASH- and ASH-driven HCC and control their response to PD-L1 blockade; 4) determine whether excessive peritumoral fibrosis correlates with diminished response to PD-1 blockade in HCC patients; and 5) determine whether agents that inhibit or attenuate stellate cell activation potentiate the response to PD- 1/PD-L1 blockade in non-viral HCC. The successful completion of these studies will result in substantial improvements to HCC immunotherapy and will establish reliable procedures for identification of patients who are most likely to benefit from PD-1/PD-L1 targeting drugs, advances that will result in significant cost savings which may amount to hundreds of millions of dollars annually.

PROJECT SUMMARY This application, ?Highly penetrant and immunogenic mouse models of non-viral HCC that are suitable for evaluation of immune checkpoint inhibitors?, is submitted in response to FOA PAR-17-245 ?Research Projects to Enhance Applicability of Mammalian Models for Translational Research?. The goal of this project is to develop accurate, innovative, and immunogenic mouse models of hepatocellular carcinoma (HCC) that closely mimic the main etiologies of human non-viral HCC in their pathogenic, transcriptomic, and genomic profiles. These etiologies include non-alcoholic (NASH) and alcoholic (ASH) steatohepatitis, and type 2 diabetes (T2D). Although the current major etiologies that underlie HCC development are hepatitis virus B and C (HBV, HCV) infections, non-viral HCC is predicted to become the major form of this aggressive cancer in the US and Europe in the not too distant future. While the incidence of non-viral HCC and its associated mortality continue to grow at an alarming rate, progress in HCC treatment has been disappointingly slow. Recently, however, inhibitors of the PD-1:PD-L1 checkpoint were found to be much more effective in HCC treatment than any other targeted or non-targeted therapeutic used in the past. Nonetheless, with objective response rates around 20%, there is much room for future improvement. Such improvement depends on better understanding of how immune checkpoint inhibition leads to HCC regression and the identification of adjuvants that enhance the response to this new class of drugs. Both objectives are dependent on the availability of immunogenic mouse models of human HCC. We will bank on our recent success in developing an immunogenic mouse model of NASH-driven HCC that is responsive to PD-1/PD-L1 blockade to develop new and improved mouse models of non-viral HCC that show rapid and synchronized tumor development, making them highly useful for translational research. In addition to NASH-driven HCC, we will develop new immunogenic models of ASH-driven HCC. We will also generate a series of mouse HCC-derived cell lines that give rise to synchronized orthotopic tumors, whose growth and response to treatment can be monitored by in vivo imaging. To determine and demonstrate the human relevance of these models, they will be subjected to extensive genomic and transcriptomic characterization and immunoprofiling. The results of these analyses will be compared to the genomic and transcriptomic profiles of human HCC using innovative computational tools. The following Specific Aims will be pursued: 1) Use the MUP- uPA model of NASH driven HCC to compare the carcinogenic efficacy of different NASH-related diets; 2) Use the MUP-uPA mouse to develop new models of ASH-induced HCC that do not involve HFD feeding; 3) Compare mouse and human HCC genomic, transcriptomic, and immune profiles; and 4) Generate cell lines from the different mouse HCC models that will give rise to synchronized orthotopic tumors that are useful for drug testing.

PROJECT SUMMARY Non-alcoholic fatty liver disease (NAFLD) is a common metabolic disorder whose US incidence exceeds 30%. While NAFLD manifests as benign steatosis, in about 15-20% of patients it assumes a much more aggressive form ? non-alcoholic steatohepatitis (NASH). The switch from benign simple steatosis to NASH is poorly understood, but was suggested to depend on ER stress. Indeed, by feeding MUP-uPA mice, which are prone to liver ER stress caused by hepatocyte-specific urokinase plasminogen activator (uPA) expression, a high-fat diet (HFD) we established a faithful model of NASH that readily progresses to hepatocellular carcinoma (HCC). HFD-fed MUP-uPA mice show ER-stress-dependent and persistent activation of sterol response element binding proteins (SREBP) 1 and 2, which control de novo lipogenesis (DNL) and cholesterol biosynthesis. Notably, SREBP1 and 2 remain activated in the MUP-uPA liver without any sign of feedback inhibition, which shuts down their activity in lipid-loaded cells. DNL is also chronically elevated in NAFLD and hepatic accumulation of free cholesterol was suggested to convert simple steatosis to NASH. Investigating how liver ER stress causes persistent SREBP activation, we uncovered a previously unknown pathway in which caspase-2 (Casp2), whose expression is ER-stress-inducible, leads to constitutive activation of site 1 protease (S1P), thereby initiating SCAP-independent SREBP cleavage in the ER. Casp2-mediated cleavage and activation of S1P seems to occur in NASH patients and genetic ablation or pharmacological inhibition of Casp2 in MUP-uPA mice blocks hepatic steatosis and NASH development. To fully establish the mechanistic aspects of this pathway and its role in hepatic steatosis, we will investigate whether it functions in liver-specific SCAP knockout mice and determine whether liver-specific Casp2 ablation affects peripheral adiposity and improves energy expenditure in addition to preventing HFD-induced hepatic steatosis and its progression to NASH. We will also generate knockin mice that express a Casp2-resistant (C2R) form of S1P and determine whether they are protected from ER- stress induced hepatic steatosis and adipose tissue expansion. As ER-stress-mediated Casp2 activation and S1P cleavage seem to depend on Tp53 activity, which results in induction of the Casp2 activator PIDD, we will examine the role of Tp53 and PIDD in Casp2-dependent S1P cleavage, SREBP1/2 activation, and hepatic steatosis, thereby establishing a key metabolic function for Tp53 outside of cancer. Finally, we will examine the hypothesis that the original function of the Casp2-dependent S1P-SREBP activation pathway was to promote energy storage in the form of liver fat during periods of hypernutrition in preparation for long-term starvation. While clarifying the role of Casp2-mediated SREBP activation in hepatic steatosis and NASH, these studies will shed new light on the poorly understood phenomenon of selective insulin resistance, in which hepatic DNL remains elevated despite diminished insulin signaling.