Ourania M. Andrisani - US grants

The long term objective of this proposal is to analyze the structural requirements of the transactivator protein CREB, employing the somatostatin promoter as the test system. The proposed studies address three aspects of the structure/function of CREB. 1. The mechanism by which CREB effects basal level transcription, by interacting with the general transcriptional apparatus. The approach involves the determination of the domain(s) of CREB required for basal level transcription, employing site directed N-terminal deletions of CREB. Analysis of the transcriptional activity of the mutant CREB proteins will be carried out by functional in vitro transcription assays. The interactions between the CREB protein mutants and the general transcription factor(s) (TFIID/TFIIB) will be assessed by functional in vitro transcription assays, complemented with recombinant TFIIB/TFIID. 2. A specific phosphorylation reaction of the CREB isoforms will be examined; namely, the sequential phosphorylation of CREB by the cAMP dependent protein kinase A and glycogen synthase kinase-3. The functional role of this sequential phosphorylation reaction will be examined by in vitro and in vivo assays. 3. The third aspect of this proposal is designed to understand how the CREB protein recognizes and interacts with the cognate CRE site. Circular dichroism and 2D-NMR studies will be utilized to examine how the Bzip domain of a recombinant CREB 259-327 peptide interacts in solution with the CRE motif.

Oncogenic viruses have been invaluable in defining cellular regulatory networks involved in cell growth control. The Hepatitis B virus X protein (pX), implicated in hepatocarcinogenesis, is a dual activator of transcription; pX activates the ras-raf-MAPK and JNK pathways, and by direct interaction, pX increases the transcriptional efficacy of bZip transcription factors, including CREB, ATF2 and ATF3. CREB mediates the transcriptional response of cAMP, is the downstream effector of mitogenic pathways, regulates c-fos expression, and is essential in development. ATF2 is also essential in development and the downstream effector of the stress-activated pathways. ATF3 is expressed in regenerating liver, is induced in response to stress and in EIA-transformed cells. Accordingly, our hypothesis is that these bZip proteins mediate cellular effects of pX-induced hepatocarcinogenesis. Our long-term goal is to define structural and functional aspects of CREB/ATF/pX interactions. Aim 1 examines the mechanism of CREB(bZip)/pX interactions by delineating a minimal, functional pX region required for CREB(bZip) interaction. Aims 2 and 3 examine the importance of CREB and ATF2 in pX-mediated transformation and the functional significance of CREB/ATF/pX interactions in hepatocarcinogenesis. These aims will be addressed employing conditional, pX-expressing, immortalized hepatocyte cell lines in which pX expression is linked to oncogenic transformation. Our studies will define: Aim 2: the mitogenic pathways activated by pX during transformation and cellular genes deregulated during pX-induced transformation; Aim 3: the role of CREB and ATF2 in pX-mediated transformation: a) by constructing cell lines in which the activity of CREB and ATF2 is attenuated; and b) by examining the role of pX in the nucleus during cellular transformation. These studies will elucidate the mechanism of CREB(bZip)/pX interactions, the role of CREB/ATF proteins in growth control in hepatocytes, and provide insights relevant to pX-mediated hepatocarcinogenesis. Studies on the mechanism of altered gene expression by pX are likely to yield important insights into the general process of oncogenic transformation in hepatocytes.

DESCRIPTION (provided by applicant): The long-term goal of the proposed studies addresses the interplay of the cAMP and BMP-2 signaling pathways in determining the differentiation of Neural Crest (NC) cells to the sympathoadrenal (SA) lineage, employing primary NC cultures. In separate studies, bone morphogenetic proteins (BMP-2, 4 and 7) and cAMP elevating agents were shown to stimulate SA cell development, characterized by the expression of tyrosine hydroxylase (TH), dopamineB-hydroxylase (DBH) and the synthesis of catecholamines (CA). Only recently, studies from our laboratory demonstrated that the cAMP signaling pathway modulates both positive and negative signals which influence SA cell development. Specifically, moderate activation of cAMP signaling, in synergy with BMP-2, promotes SA cell development and induces the expression of the SA lineage-determining gene Phox2a. By contrast, robust activation of cAMP signaling opposes, even in the presence of BMP-2, SA cell development and blocks the expression of the SA lineage-determining genes ASH-I and Phox2a. The objectives of the proposed studies investigate the molecular mechanisms by which cAMP acts as a bimodal switch on SA cell development. Aim 1 addresses the hypothesis that low-level cAMP signaling synergizes transcriptionally with BMP-2 signaling at the phox2a gene promoter, via synergistic interactions occurring between the transcription factors ASH-i and CREB, and the co-activator protein CBP. Aim 2 addresses the hypothesis that the antagonistic effect of high-level cAMP signaling on SA cell development involves the activation of the MAPK pathway, effecting the inhibition of the BMP-2-activated transactivators SMADs. The Specific Aims are: Aim 1: To determine whether the synergistic effect between BMP-2 and low-level activation of cAMP signaling, on SA cell development and Phox2a expression, is linked to the transcriptional function of the cAMP pathway. Aim 2: To define the mechanism of the antagonistic effect of high-level activation of cAMP signaling on the BMP-2 mediated ASH-I gene expression and SA cell development.

[unreadable] DESCRIPTION (provided by applicant): CREB/ATF proteins, the effectors of the mitogenic ras-raf-MAPK, JNK and p38MAPK pathways, mediate gene expression required for proliferation, response to stress, differentiation and apoptosis. CREB/ATF proteins, via the bZip domain, interact with Hepatitis B virus (HBV) X protein (pX), implicated in hepatocellular carcinoma (HCC) development. Since viral on co-proteins effect transformation by deregulating key, cellular growth control mechanisms, we hypothesize that pX interaction with CREB/ATF is important in hepatocyte transformation. In the previous funding period we have tested specific aspects of this hypothesis and have: 1) delineated the minimal region of pX required for interaction with CREB/ATF proteins; and 2) developed and characterized a novel, comparative in vitro cellular model of pX-mediated hepatocarcinogenesis. This cellular model is comprised of two tetracycline-regulated, pX-expressing cell lines derived from the AML12immortalized hepatocyte cell line. The 3pX-1 cell line is a differentiated hepatocyte that becomes transformed by pX; the other, 4pX-1, is a de-differentiated hepatocyte cell line that does not display pX-mediated transformation, but is sensitive to pX-mediated apoptosis. The goal of this proposal is to gain better understanding of the mechanism of pX-mediated hepatocyte transformation, and the role of CREB/ATF proteins in pX-mediated hepatocyte transformation and apoptosis. Since our earliest studies defined the minimal pX region required for increased CREB/ATF transcriptional efficacy, in Aim 1 we will delineate the minimal pX region required for transformation in differentiated hepatocytes vs. apoptosis in de-differentiated hepatocytes. In Aim 2 we will investigate the mechanism by which pX sensitizes de-differentiated 4pX-1 cells to apoptosis, and the pX-mediated mechanism(s) that rescue 4pX-1 cells from apoptosis, resulting in transformed hepatocytes. Thus, we will investigate the hypothesis that the de-differentiated 4pX-1 cells model a precancerous precursor for HCC. In Aim 3 we will characterize two cloned, novel ESTs expressed during pX-mediated hepatocyte transformation. The proposed studies will elucidate further the mechanism of pox-mediated transformation; the mechanism of pX mediated apoptosis, and the cellular precancerous precursor of HCC; and will characterize new molecules, which have the potential of being early diagnostic markers in human HCC development. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): We are requesting support for an innovative, new program to educate the next generation of research veterinarians in whole animal-based biomedical sciences and hypothesis-driven research. The Purdue University School of Veterinary Medicine (PU-SVM) has long recognized the importance of interfacing the education of its graduates in veterinary medicine with hypothesis-driven basic biomedical research. To this end the PU-SVM has implemented this interfacing of professional and basic biomedical research education. Since 1991, 85 veterinary students have participated in summer research programs in PU-SVM. Seventeen percent of these veterinary graduates are in careers linking directly veterinary medicine and biomedical sciences, or are now pursuing advanced graduate degrees. The long-term goal of the PU-SVM, with support from NIH, is to expand the scope of the existing program, enabling qualified veterinary students to complete both their professional DVM degree and a degree(s) in basic biomedical sciences, with a primary focus in either cancer or neuroscience research. We propose to develop a dual degree DVM/Masters program by: a) targeting recruitment of third year veterinary students for one-year formal training program in biomedical research, b) enabling these students to concurrently complete their DVM degree and graduate Master's degree in Basic Medical Sciences. This proposed dual DVM/MS degree program builds upon the flexibility of the tracked fourth-year curriculum in our school. The positive outcomes of this integrated professional and graduate education will be the next generation of veterinary scientists, who will be able to participate in clinically relevant investigations, employing emerging basic science knowledge, and/or in basic biomedical investigations, employing a clinical perspective and designed to improve the health of both animals and humans. In addition to implementing the Integrated Veterinary - Biomedical Research Training (IVBMRT) program, we will continue to provide opportunity for veterinary students, who have completed the first or second years of the DVM curriculum and want an animal-based, hypothesis-driven biomedical research experience without entering a graduate degree program. Participating faculty mentors in the proposed IVBMRT program include faculty from the PU-SVM, the Schools of Pharmacy and Science and Liberal Arts, thus linking the medical perspective of the SVM with the fundamental biomedical perspectives and expertise also present at Purdue University. Importantly, these Purdue faculty mentors together represent a diversity of ethnic groups and national origins, varied cultural backgrounds, many of the major research universities of the US and abroad, as well as minorities and women. This mentor diversity will facilitate recruitment and retention of students from minority and underrepresented groups while enriching the research education and training of all participating students.

DESCRIPTION (provided by applicant): Chronic Hepatitis B virus (HBV) infection is linked to hepatocellular carcinoma (HCC). The HBV X protein (pX) is implicated in HCC pathogenesis by an unknown mechanism. The long term goals of this study are to determine how pX initiates hepatocyte transformation and to identify new targets for therapy. Our previous studies have found that in non-transformed hepatocytes, pX induces DNA re-replication, resulting in DNA damage and polyploidy. As a result, the replication stress kinase ATR is activated which is known to activate p53. However, despite DNA damage, pX-expressing hepatocytes do not die and instead proceed to mitosis, where DNA damage is propagated to daughter cells, giving rise to polyploidy. How this process occurs is unknown and is critical for understanding both pX-induced oncogenic transformation and the cellular mechanisms involved in maintaining genomic integrity. To identify molecules involved with these mechanisms, we employed a lentiviral siRNA library and identified genes whose depletion rescues pX-expressing cells from DNA damage-induced apoptosis. We identified genes involved in 1) DNA replication and mitotic progression, 2) DNA repair and 3) p53 function. Accordingly, the main objective of this proposal is to determine how pX deregulates these mechanisms, resulting in oncogenic transformation. We hypothesize that a likely candidate for pX deregulation is Polo-like kinase1 (Plk1) because some of the depleted genes (e.g., mitotic progression genes) are known Plk1 substrates. In addition, Plk1 terminates the G2/M DNA damage checkpoint to initiate mitosis, and importantly, we have found that pX induces both Plk1 expression and activity. Significantly, Plk1 is overexpressed in liver tumor samples from HBV-HCC patients. Since pX induces Plk1 activity, our hypothesis is that Plk1 terminates not only the G2/M DNA damage checkpoint of pX-expressing hepatocytes, but also suppresses DNA repair and the pro-apoptotic function of p53. Consequently, pX-expressing cells with DNA damage escape apoptosis continue to accrue DNA damage and suffer from genomic instability leading to malignant transformation. To address this hypothesis, we will investigate the G2 phase of X-expressing cells and establish the role of Plk1 in terminating the DNA damage checkpoint (Aim 1), the role of Plk1 in termination of DNA repair and p53 apoptosis (Aim 2), and the mechanism by which Plk1 terminates DNA repair and p53 transcription (Aim 3). Significance: We will investigate the role of Plk1 in HBV pX-mediated hepatocyte transformation. Plk1 is overexpressed in human liver tumors but its direct link to liver cancer pathogenesis is unknown. Our studies will link Plk1 to oncogenic transformation and explore novel functions of Plk1 in DNA repair and p53 apoptosis. Significantly, Plk1 inhibition selectively kills tumor cell lines and a Plk1 inhibitor is currently in clinical trials. Given the magnitude of chronic HBV infection worldwide and that HBV-HCC is usually fatal, our studies have the potential to identify Plk1 as a new diagnostic marker and a therapeutic target for HBV-HCC. PUBLIC HEALTH RELEVANCE: Chronic HBV infection leads to fatal liver cancer (1). The World Health Organization reports 400 million people are chronically infected with HBV, placing them at a greatly increased risk for HCC development. Thus, studies of how HBV causes liver cancer address a significant human health problem needing new and efficacious therapies.

DESCRIPTION (provided by applicant): Chronic Hepatitis B Virus (HBV) infection is a major factor in the pathogenesis of hepatocellular carcinoma (HCC). Despite the HBV vaccine, the World Health Organization reports 400 million people are chronically infected with HBV. Current treatments are ineffective, and rates of primary HCC have tripled in the U.S. from 1975-2005. To reduce liver cancer, new and effective therapies are needed, as well as new biomarkers for molecular classification and prognosis of the disease. In this proposal we investigate the role of the mitotic Polo-like kinase1 (Plk1) in the pathogenesis of HBV-mediated liver cancer (HBV-HCC). Pathogenesis of HBV-HCC involves chronic liver inflammation and effects of the weakly oncogenic HBV X protein (pX). pX activates cellular mitogenic pathways, promotes DNA re-replication-induced DNA damage and activates Plk1. In turn, Plk1 mediates checkpoint adaptation, generating partial polyploidy in non- transformed pX-expressing hepatocytes. Significantly, inhibition of Plk1 suppresses pX-mediated transformation. However much remains to be understood about the role of Plk1 in pX-mediated transformation and HBV-HCC. Toward this end, we have identified by a genome-wide siRNA library screen, SUZ12 and ZNF198 as tumor suppressors of pX-mediated transformation. Our results indicate that these proteins are negatively regulated by Plk1. Both in human liver cancer cell lines and tissues from chronic HBV patients with HCC, protein levels of Plk1 are increased, whereas those of SUZ12 and ZNF198 are reduced relative to normal controls. This inverse relationship (high protein levels of Plk1 and reduced levels of SUZ12 & ZNF198) also occurs during HBV replication, suggesting overexpression of Plk1 and down-regulation of SUZ12 & ZNF198 is important both for transformation and HBV replication. SUZ12 and ZNF198 mediate chromatin remodeling and associate with PML nuclear bodies (NBs) that regulate DNA repair, apoptosis and viral replication. We reason down-regulation of SUZ12 & ZNF198 by Plk1 alters hepatocyte gene expression and disrupts the function of PML NBs. Accordingly, our hypothesis is: Plk1 down-regulates SUZ12 and ZNF198, which enhances HBV replication by disrupting PML NBs, and mediates oncogenic transformation by deregulating hepatocyte gene expression. We will investigate in Aim 1, the role of Plk1, SUZ12, and ZNF198 in pX-mediated transformation and HBV replication; in Aim 2, the mechanism by which Plk1 down-regulates ZNF198 and SUZ12. In Aim 3, we will investigate whether protein levels of Plk1, SUZ12, ZNF198, and expression of known SUZ12 target genes are prognostic for disease progression and survival. Impact: High level of viremia in chronic HBV patients is a risk factor for progression to HCC. Inhibition of Plk1 could serve as a therapy strategy to suppress HBV replication, reducing the risk of HCC development. Plk1 inhibitors are in clinical trials for other types of cancer and could serve as therapy for HBV-HCC. Our studies hold promise to reveal novel therapy targets and biomarkers for HBV-HCC.

ABSTRACT Chronic Hepatitis B Virus (HBV) infection is a major risk factor in development of hepatocellular carcinoma (HCC). Despite the HBV vaccine, the World Health Organization (WHO) reports 250 million people are chronically infected with HBV. Current therapies for HBV infection or treatment of liver cancer are ineffective. To reduce the risk for liver cancer, new and effective therapies are needed, targeting essential mechanisms of viral replication and liver cancer pathogenesis. In this proposal we are investigating a novel epigenetic mechanism that contributes both to HBV biosynthesis and HBV-associated liver cancer. This mechanism involves the chromatin modifying Polycomb Repressive Complex 2 (PRC2) that silences genes by H3K27 trimethylation, the DEAD box helicase DDX5 that remodels RNA protein (RNP) complexes, and the long noncoding RNA (lncRNA) HOTAIR. PRC2 silences transcription of >1000 genes and binds >9,000 lncRNAs, including HOTAIR. However, how PRC2 targets repression of specific genes is not yet understood. Our studies have identified the RNA helicase DDX5 as a regulator of PRC2-mediated gene repression, acting by stabilizing the essential PRC2 subunit SUZ12, via regulation of RNP complexes formed with HOTAIR. Significantly, knockdown of either DDX5 and/or HOTAIR enabled re-expression of specific PRC2-repressed genes, namely, EpCAM and pluripotency genes (NANOG, OCT4, and SOX2.) Concerning the role of this epigenetic mechanism in HBV infection and HCC pathogenesis, we have shown the HBV encoded X protein, a cofactor in hepatocarcinogenesis, signals the proteasomal degradation of SUZ12 protein. The downregulation of SUZ12 results in loss of PRC2 function and re-expression of EpCAM and pluripotency genes during HBV replication and in HBV-associated liver tumors. In addition to SUZ12, we have also found that DDX5 is downregulated during HBV replication and in poor prognosis HBV-induced HCCs by an unknown mechanism. Importantly, downregulation of SUZ12 and DDX5 is advantageous to virus biosynthesis. In this competitive renewal application, our working hypothesis is that DDX5 functions to stabilize SUZ12 and the PRC2/HOTAIR complex, thus promoting PRC2-mediated transcriptional repression of both cellular and viral genes. How viral infection deregulates this mechanism is not understood. Accordingly we will investigate: in Aim1, the role of DDX5 in SUZ12/PRC2 function, and how HBV infection deregulates this complex; in Aim2 the role of DDX5 in HBV replication, and in Aim3 the mechanism of DDX5 down-regulation during HBV infection, and its role in HBV-mediated tumorigenesis. Impact: The proposed studies will elucidate a novel epigenetic mechanism that regulates both HBV biosynthesis and HBV-mediated oncogenic transformation. Our studies have the potential to identify novel therapy targets for HBV infection and liver cancer, e.g., the RNA helicase DDX5 and molecules that regulate DDX5.

Cell Identity and Signaling (CIS) Research Program Project Summary The key scientific goals of the Cell Identity and Signaling (CIS) Research Program are to advance discovery of novel molecular mechanisms of cell identity and cell signaling, to apply this knowledge towards understanding cancer pathogenesis, and to use this knowledge to develop novel, mechanism-based approaches to prevent or interfere with cancer cell growth, aiming for cancer solutions. It is well-established that cancer cells hijack normal regulation of cell growth, differentiation, and embryonic development, via genetic and epigenetic mechanisms. The CIS Program aims to understand these fundamental mechanisms and shepherd them toward cancer solutions. The CIS Program has 27 members, $5.5 million in cancer-focused, peer-reviewed extramural funding, with 38% of the total funding from the NCI. CIS research themes span a spectrum from basic discovery, using simple model organisms and cellular and animal cancer models, to cancer solutions. CIS members are highly productive with 202 cancer-related publications since July 2015, and highly interactive with a 70% increase in collaborative publications. Importantly, 73% of all cancer-relevant CIS publications are collaborative. In the previous funding cycle, the CIS Program, supported by competitive pilot grants from the Purdue Center for Cancer Research (PCCR), successfully fostered highly collaborative, cancer-relevant studies linking CIS Program themes (intra- programmatic) with other PCCR programs (inter-programmatic), and also with external partners (inter- institutional). For the next funding period, the goal of the CIS Program is to advance the breadth and depth of our understanding of cancer-relevant mechanisms and to maximize their transition to cancer solutions. The approach towards this goal is to enable and foster collaborative and transdisciplinary studies by providing competitive PCCR pilot grants, and access to state-of-the-art, PCCR-supported Shared Resources, and modern technology in structural biology, drug discovery, cancer genomics, bioinformatics and computational biology. Three specific aims are proposed. Aim 1: To further enhance discovery of basic and cancer-relevant mechanisms by strengthening the integration of computational genomics and bioinformatics and increasing expertise and training in computational biology. Aim 2: To enhance discovery of cancer-relevant mechanisms of signal transduction, gene expression and epigenetics by supporting collaborative, transdisciplinary approaches and modern technologies. Aim 3: To accelerate transition of newly discovered cancer-relevant mechanisms towards cancer solutions, by developing essential mechanisms as therapy targets, and by employing transdisciplinary approaches.

ABSTRACT/SUMMARY Hepatitis B virus (HBV) infection is a significant global public health problem, with over 290 million people worldwide being chronically infected. Significantly, chronic HBV infection is associated with increased risk for development of hepatocellular carcinoma (HCC), the 2nd leading cause of cancer-related mortality worldwide. Despite the effectiveness of the prophylactic HBV vaccine, there is no cure for chronic HBV infection or the resultant liver cancer. There is compelling need to discover new therapeutic strategies to cure chronic HBV infection and HCC pathogenesis. Thus, the global HBV scientific community must continue to make directed strides towards understanding key, unanswered aspects of HBV biosynthesis and disease pathogenesis. The annual meeting on the Molecular Biology of Hepatitis B Viruses is the only forum that brings together the international community of scientists who study mechanisms of biosynthesis and pathogenesis by HBV and and its closely associated hepatitis delta virus (HDV). This International meeting on Hepatitis B Viruses has been held yearly since 1985, alternating at sites in North America, Europe, Asia and Australia. In 2020, the meeting will be held in Toronto, Canada. The success and achievements of the HBV field relies on the contributions of not only the established investigators but more importantly, on early career researchers (ECRs) that have/are deciding to enter the field, bringing with them new technology and concepts. Accordingly, the Specific Aim of this R13 application is: to promote and maximize attendance and participation of early career researchers/ECRs (graduate students & postdoctoral fellows) at the 2020 International HBV Meeting. The 2020 meeting will provide the stage for scientific exchange and dissemination of the latest research results that could influence new therapeutic strategies for HBV and HDV infections. It is estimated that 500-600 delegates will attend the 2020 meeting. The organization of the meeting will consist of 8 oral scientific sessions and 5 poster sessions including for the first time, e-poster presentations, as a training stage for early career scientists. In support of the goal of the global scientific strategy to cure HBV, keynote speakers will present new perspectives and technologies, and discuss standards that can be applied to advance the HBV field. To improve scientific interactions and scientific exchange, social gatherings are organized at the end of each meeting day. Significantly, since 2005, the International HBV meeting enjoys the continuing support from the Hepatitis B Foundation, minimizing the meeting costs, and publicizing the meeting to universities with large numbers of underrepresented minorities. To enable participation of early career trainees and underrepresented minority investigators, support from the National Institutes of Health to help defray the travel costs is requested. This funding will be used to ensure attendance and presentations by early career researchers, particularly from countries with lesser developed research capacity, as well as underrepresented minority investigators.