Raul A. Urrutia - US grants

Proper functioning of the mammalian nervous system depends on a specific wiring pattern determined by a developmental program. This program directs how neurons divide at early stages, migrate to their final destination, outgrow cell processes and, finally, establish synapses with other neurons or a distinct target cell. A significant number of human diseases occur as a result of an aberrant completion of this developmental program. Many of these diseases, including Down's Syndrome, several forms of congenital mental retardation, phenylketonuria, malnutrition and schizophrenia are characterized by neurons with an alteration in the normal structure and number of neurites. Microtubules and microtubule-associated proteins and enzymes are known to play a crucial role in the development of normal wiring patterns in the nervous system by taking part in the extension of neuronal processes. Our laboratory is focused on the role of a specific microtubule-associated enzyme, dynamin, in the extension of neuronal processes. Dynamin is a microtubule-activated GTPase that binds and cross-links microtubules in vitro in a nucleotide-dependent manner and therefore has been proposed to act as a microtubule-based motor in vivo. We have made important preliminary observations which indicate that 1) dynamin is essential for neurite formation and 2) dynamin is localized at the neuronal growth cone. In addition, we have cloned a novel dynamin gene encoding a second isoform expressed in rat brain. From these studies, we hypothesize that dynamin contributes to the formation of neurites during development by: a) maintaining the normal structure of neuronal processes through cross-linking and positioning microtubules in the neurite and b) participating in the endocytic recycling of proteins which are important for neurite outgrowth. We have designed experiments to correlate the level of dynamin with the formation of neurites in both hippocampal neurons and PC12 cells developing in vitro. Furthermore, additional studies are aimed to alter the expression of dynamin using antisense technology and determine whether a reduced level of this protein impairs neurite outgrowth. We will use state-of-the-art video and electron microscopy in combination with molecular biological techniques to determine the integrity of the microtubule network and endocytic vesicular transport pathway in the dynamin minus neurons. To our knowledge, this is the first study which has provided insight into the in vitro role of dynamin in neuronal development. We are optimistic that a detailed characterization of the mechanisms involved in neurite formation will help us better understand many mental diseases.

Our long-term goal is to define molecular mechanisms important for the regulation of cell proliferation and apoptosis in the exocrine pancreas which is a crucial step for better understanding normal morphogenesis and pancreatic cancer. Toward this end, we are studying the role of growth-factor inducible C2H2 zinc finger transcription factors in the regulation of these phenomena. Zinc finger proteins play a crucial role in organogenesis in several mammalian tissues, and mutations in some of these genes give rise to neoplastic transformation. However, their presence and function in the exocrine pancreas remain to be elucidated. The mechanistic experiments outlined in this proposal will test the central hypothesis that a novel TGFbeta-inducible zinc finger protein TIEG is a transcription factor involved in the regulation of apoptosis and/or the cell cycle in pancreatic cells. We have isolated a TIEG cDNA from a rat pancreas library and demonstrated that this gene is an early response target for TGFbeta in exocrine pancreatic cell populations. Interestingly, although its biochemical properties have not yet been determined, sequence analysis of the deduced TIEG protein reveals the presence of several motifs that are characteristic of transcription factors. Because TGFbeta induces both apoptosis and cell cycle arrest in pancreatic cell populations, TIEG is a good candidate to participate in these phenomena. Indeed, we have recently shown that the overexpression of TIEG in pancreatic cell populations induces apoptosis. Thus, in this proposal, we hypothesize that: 1) TIEG functions as a sequence-specific transcription factor, 2) the apoptotic effects of TIEG depend on the activity of this protein as a transcription factor, and 3) the overexpression of TIEG induces cell cycle arrest prior to apoptosis. These hypotheses will be addressed in the following specific aims: 1) Determine the nuclear localization and transcriptional regulatory activity of TIEG, 2) Determine the DNA binding sequence(s) for TIEG, and 3) Characterize the mechanisms involved in TIEG-induced apoptosis (transcriptional activity and cell cycle arrest). We propose to use state-of-the-art molecular techniques in combination with well- established functional assays to approach these aims. We are optimistic that the successful completion of this proposal will significantly advance our understanding on the role of zinc finger proteins in pancreatic cell physiology and begin to fill a gap in the existing knowledge in this underrepresented area of pancreatic research. Furthermore, this information will be crucial as a theoretical framework for future studies on the role of zinc finger transcription factors in pancreatic development and cancer.

Zinc finger proteins are key regulators of normal morphogenesisi and can also act as either oncogenes or tumor suppressors. The investigators laboratory has been focused on studying the role of these proteins in the regulation of pancreatic gene expression, cell growth, and the modulation of neoplastic transformation. Pancreatic cancer currently ranks fifth as a cause of death by cancer in the USA and has one fo the poorest prognoses among the human neoplasias. Although the etiology and pathophysiology of pancreatic ductal cancer is poorly understood, increasing evidence indicates that alterations in transcription factors (e.g. DPC4/Smad4) are often associated with this disease. Furthermore, transcription factors such as p53 are beginning to be tested as genetic therapeutic tools to treat this disease. The proposal is focused on the biochemical and functional characterization of KS1, a novel zinc finger transcription factor isolated from the pancreas that represses transcription and suppresses neoplastic transformation. Both of these functional properties of KS1 are interrelated since the deletion of a distinct transcriptional repressor domain, R2 abolishes the ability of this protein to suppress neoplastic transformation. Currently, however, the mechanisms that KS1 uses to perform these functions are poorly understood. Moreover, the role of KS1 in the regulation of pancreatic epithelial cell growth is also undefined. In this proposal, the investigator will test the central hypothesis that KS1 is a sequence-specific transcriptional repressor protein that inhibits cell growth and neoplastic transformation by regulating apoptosis and/or cell proliferation. The investigator will use state-of-the-art biochemical and molecular techniques combined with well established assays and cell models for studying neoplastic transformation and pancreatic epithelial cell growth. The proposed aims are to: 1) determine the role of KS1 in the regulation of cell proliferation and apoptosis, 2) determine the DNA binding activity of KS1, and 3) characterize the mechanisms underlying the function of the R2 domain of KS1 as a transcriptional repressor and suppressor of transformation. The investigator expects that successful completion of this proposal will provide novel information on molecular mechanisms used by zinc finger proteins to regulate cell growth and neoplastic transformation. This knowledge will serve as the foundation for future studies aimed at using these proteins as therapeutic tools for controlling abnormal cell growth.

DESCRIPTION (provided by applicant): Our long term goal is to discover mechanisms that mediate silencing of tumor suppressor pathways during the development of pancreatic cancer, the 4th leading cause of death by cancer in USA and an extremely painful disease. Congruent with this goal, this proposal seeks to define basic mechanisms underlying the regulation of exocrine pancreatic cell growth by novel TGF[unreadable]-inducible KLF zinc finger repressor proteins discovered by our laboratory. KLF proteins have recently elicited significant interest because of their role in regulating gene expression, normal morphogenesis, and neoplastic transformation. Our previous observations led us to discoveries in three important areas: 1) cell growth suppression, 2) transcriptional repression, and 3) TGF[unreadable] ?signaling. Now, our preliminary data supports a unifying theme by which these three areas may be linked, namely studies on novel coregulator molecules for KLF11 (the structural and functional paradigm of this subfamily of TGF[unreadable]-inducible KLF repressors) and their role in modulating the expression of members of the TGF[unreadable] superfamily of signaling molecules. We chose this focus because of its high potential for providing novel mechanistic information that can fuel advances in our field of study. Corepressors constitute a new and exciting area of research which recently exploded after the discovery of histone-deacetylases, methylases, and chromatin remodeling machines in mammalian cells. Few corepressors are known for KLF proteins, and rudimentary knowledge is available on their role in the regulation of exocrine pancreatic cell growth and diseases. Thus our central hypothesis is that novel protein interactions modulate the function of KLF11 in gene expression and/or cell growth regulation, as well as neoplastic transformation via the regulation of members from the TGF[unreadable] ?superfamily of signaling molecules. Our aims will test the following hypothesis: Aim 1: Interaction with HP1 modulates the ability of KLF11 to regulate gene expression, cell growth, and neoplastic transformation;Aim 2: Interaction with the G protein [unreadable] subunit modulates the ability of KLF11 to regulate gene expression, cell growth, and neoplastic transformation, and;Aim 3: KLF11 regulates the expression of targets from the TGF[unreadable] family of signaling proteins known to be involved in the regulation of normal cell growth and/or neoplastic transformation. These experiments will use state-of-the-art cellular and molecular techniques for analyzing both transcriptional repression and cell growth. We believe that linking corepressors with cell growth regulation and TGF[unreadable] signaling makes this proposal innovative, hypothesis driven, highly focused, biologically and medically relevant, and feasible taking into consideration our expertise, previous published work, and current preliminary data. We are optimistic successful completion of these studies will help to build a useful theoretical framework for better understanding morphogenetic pathways that are active in exocrine pancreatic cells and help to maintain their homeostasis, regulate morphogenesis, and modulate neoplastic transformation. PUBLIC HEALTH RELEVANCE: These investigations represent a defined strategy to discover how pancreatic cancer, a painful and deadly disease that ranks 4th as the cause of death by cancer in USA, arises. Patients affected by this cancer die within 3 to 6 months after the diagnosis, and currently, no effective treatment exists for this disease. However, we are optimistic that our studies will build the foundation for future treatment and perhaps even the prevention of this disease.

DESCRIPTION (provided by applicant): This proposal requests the renewal of our major source of long-standing funding for investigating the epigenetics of pancreatic diseases. Our studies will directly extend our knowledge on common diseases, such as chronic pancreatitis and pancreatic ductal adenocarcinoma (PDAC), both painful and incurable disorders of the exocrine pancreas for which effective treatments remain to be discovered. Our OVERALL OBJECTIVE is to unravel novel epigenetic mechanisms that extend oncogenic growth signals downstream from KRAS during initiation and pancreatitis-associated cancer promotion. Our preliminary data identify HP1? as a pro-oncogenic epigenetic regulator of gene activation and growth, which is overexpressed in human pancreatic cancer as well as in animal models of pancreatic carcinogenesis. We provide solid evidence that this increased level of HP1? enhances the malignant effects of the KRAS oncogene. Our CENTRAL HYPOTHESIS is that the epigenetic regulator, HP1, works downstream from KRAS to promote pancreatic cell growth by regulating the expression of proliferative gene networks induced by this oncogene. Our AIMS will test the following hypotheses: Aim 1: HP1? participates in a membrane-to-nucleus gene regulatory pathway that enhances the ability of KRAS to mediate neoplastic transformation and tumorigenesis; Aim 2: HP1? works downstream of KRAS to regulate growth-promoting gene networks; and Aim 3: HP1? inhibition ameliorates KrasG12D-mediated PDAC initiation and promotion after pancreatitis. Our design proposes molecular, cellular, and whole organism experiments using state-of-the-art methodologies. Accordingly our laboratory has developed the appropriate conceptual framework, reagents, trained personnel and has established productive collaborations. The innovative design of this proposal seeks to maximize the yield of mechanistic and rapidly translatable knowledge in this underepresented yet extremely the new promising area of epigenetics in pancreatic diseases. As new drugs targeting both the KRAS and HP1 pathways are being tested in clinical trials, this proposal builds the rationale for applying these tools to the management of patients affected with deadly pancreatic diseases, thereby bearing significant biomedical relevance.

PROJECT SUMMARY This objective of this Research Supplement to Promote Diversity in Health-Related Research (PA-21-071) is to provide a mentored experience in basic and translational science for Dr. Callisia N. Clarke. The purpose of the parent R01 (DK052913; Raul Urrutia, MD; contact PI; and Gwen Lomberk, PhD; PI) is to investigate the role of epigenetic regulation in pathophysiological mechanisms underlying pancreatic diseases and to understand how potentially druggable epigenomic landscapes, identified from patient-derived material, are linked to development of preneoplastic and neoplastic diseases of the pancreas. Cell growth regulation that is vital for pancreatic development and homeostasis is altered in most pancreatic disease states, including cystic lesions, IPMN, and pancreatic cancer. Research conducted in the mentors' laboratories on patient derived tissue encompasses a multi-parametric integrative analysis of ChIP-seq on multiple histone modifications, RNA-seq, and DNA methylation to define epigenomic signatures for human pancreatic ductal adenocarcinoma (PDAC) subtypes, which can predict their relative aggressiveness and survival[1]. However, little is known about the impact of epigenetic regulation on the development and progression of pancreatic neuroendocrine tumors (PNET). PNETs are rare tumors with significant heterogeneity in clinical presentation and prognosis. Functional PNET (F-PNET) are associated with hormone secretion causing a clinical syndrome, while non-functional (NF-PNET) tumors are not associated with hormone-driven symptoms. This supplemental application is designed to expand studies of the parent grant beyond PDAC and characterize modifications in gene expression mediated by noncoding RNA (ncRNA) in PNET. Our Central Hypothesis is that epigenetic mechanisms, at the level of ncRNA, lead to silencing of the functional phenotype of PNET, yielding NF-PNET. Our specific aims are: 1. To determine the distinct epigenomic signatures that exist between F-PNET and NF-PNET using ncRNA sequencing analysis and 2. To evaluate the impact of ncRNA signatures on NF-PNET treatment response, disease progression, and patient survival. Dr. Clarke will be mentored by an outstanding team of investigators with complementary expertise in the areas of epigenomics and bioinformatics. The mentoring/career development plan will focus on research design, methodology, and data analysis. The proposed research aligns with the parent R01 study in conceptual framework and necessary expertise. Furthermore, this Research Supplement will help in our commitment to promote the growth of one of our minority researchers in health sciences. Results from this study will serve as preliminary data for a NIH K08 award. Dr. Clarke's long-term goal is to develop her independent research program in PNET tumorigenesis and to identify potentially actionable epigenetic pathways to interrupt progression of this disease.