J. Thomas Parsons - US grants

Genetic and biochemical experiments have shown that the product of the Rous sarcoma virus src gene is required for the initiation and maintenance of cellular transformation. As a part of our long-term goal of understanding how the src gene product, pp60src, mediates the molecular events involved in cellular transformation, we have isolated and partially characterized monoclonal antibodies directed against pp60src. The experiments outlined in this proposal employ monoclonal antibodies to unique determinants within pp60src as immunochemical probes to define and characterize the structural and functional domains of the molecule and attempt to elucidate the role of individual functional domains in mediating cellular transformation. We further propose to utilize monoclonal antibodies to pp60src as immunochemical probes to compare the structure and function of other retrovirus tyrosine protein kinase as well as normal cell tyrosine protein kinases. These studies will permit us to define the structural and functional domains of pp60src and allow us to relate this information to other proteins which may be members of a family of related tyrosine protein kinases.

The long term goals of the studies outlined in this proposal are to understand how tyrosine kinase onco-proteins (e.g., pp60src) and effectors of normal mitogenic responses (e.g., growth factors and their receptors) function together to influence cellular growth regulation. Oncogenic transformation mediated by pp60src induces the expression of a novel growth factor receptor, c-sea. C-sea expression is also induced in response to serum stimulation of quiescent fibroblasts, suggesting that c-sea expression may be linked to mitogen induced proliferation. We propose to study the molecular events linking pp60src expression, mitogen stimulation and expression of the c-sea growth factor receptor. In Aim 1 we will identify and characterize the c-sea gene product. The sequence analysis of chicken c-sea and cloning and sequencing of the human homologue of c-sea will be carried out. Antibodies to both extra-cellular and cytoplasmic domains of the c-sea protein will be used to identify and characterize the c-sea protein. We will use both c-sea cDNA probes and antibodies to determine the tissue and cell type distribution of c-sea expression, and investigate the cell cycle dependent expression of c-sea. In Aim 2 we will characterize the c-sea protein and assess its possible function as a receptor-like tyrosine protein kinase. We will isolate cell lines over- expressing the complete c-sea gene and various structurally permuted variants of c-sea. These cell lines will be analyzed with respect to morphological phenotype, growth factor responses and tyrosine phosphorylation of the c-sea protein and tyrosine phosphorylation of putative cellular targets for c-sea. We will attempt to activate the over- expressed c-sea receptor in a ligand-dependent manner and initiate experiments to identify the natural ligand for the c-sea receptor. In Aim 3 we will determine the role of c-sea expression in contributing to and/or mediating the transformed phenotype of src transformed cells. We will attempt to block functional expression of c-sea in src transformed cells and assess the phenotypic consequences of such a block and we will carry out studies to identify the src induced signalling pathways that lead to c- sea induction. The analysis of the c-sea receptor may provide insights into the possible altered expression of c-sea in human and animal tumors and provide information regarding the mechanisms of c-sea gene expression during the cell cycle.

The long term goals of this revised Program Project are to understand the molecular pathways that regulate cell growth and trigger specialized cell functions. Understanding these fundamental signalling pathways will contribute to our knowledge of abnormal signalling pathways operative in cancer cells. The mechanisms that control cell growth, differentiation and specialized cell functions involve the complex interactions of cellular receptors with a variety of hormones and mitogens. In addition these cellular processes are profoundly influenced by the interaction of the cell with components of the extracellular matrix, as well as with molecules present on the surface of neighboring cells. The projects outlined in this Program Project application have a common theme in that each project seeks to define and understand the role of protein phosphorylation, catalyzed by tyrosine and/or serine/threonine kinases, in the regulation of well studied and characterized signal transduction pathways. Michael Weber continues his investigation of the EGF receptor and its interactions with pp60src. T. Parsons proposes to study the role of a recently discovered tyrosine kinase, termed Focal Adhesion Kinase, in cellular signalling. J. Garrison builds on studies carried out during the past project period addressing the effects of v-src on inositol lipid signalling. Garrison proposes to explore in detail the nature of v-src stimulation of the enthothelin receptor pathway, focusing on the interactions among the receptor, the G protein, Gq and PLC beta. T. Bender will study the c-myb protein determining how protein phosphorylation regulates its activity as a transcription factor. Projects by Parsons and Crentz involve studies on the signalling pathways that control secretion events in adrenal chromaffin cells. S. Parsons will continue her studies on the role of pp60src of phosphorylation on annexin function. The Program Project will be supported by two Cores, an Administrative Core and a Scientific Core devoted to production of antibodies, productions and purification of proteins and handling of radioactive materials.

The long term goals of this revised project are to understand the nature of cellular signalling events mediated by cellular receptors that recognize components of the extracellular matrix and/or other cell surface molecules. We will focus specifically on the role of tyrosine phosphorylation in regulating these pathways, seeking to define the molecular mechanisms by which tyrosine kinases contribute to the regulation and control of such pathways. The proposed experiments build and extend the progress made during the past four years. We have identified a novel protein tyrosine kinase, that is associated with cellular focal adhesions, designated Focal Adhesion Kinase, FAK. Evidence from our own laboratory as well as other has indicated that FAK plays a role in regulating signalling events initiated by interactions of surface integrins with extracellular matrix. In addition, our own studies have demonstrated a stable association of FAK with pp60src in src transformed cells. We outline three specific aims: First, we will define and characterize the mechanisms that lead to activation of FAK in response to engagement of integrins with defined extracellular ligands. These studies will include an analysis of the interaction of FAK with cytoplasmic domains of specific integrins and components of focal adhesions as well as possible functional interactions with proteins that regulate mitogen and hormone activated signal transduction pathways. Second, we will examine the role of FAK in the regulation of the formation and/or breakdown of cellular focal adhesions, in the regulation of other cellular activities (e.g., adhesion, cell spreading, cell migration) and the possible control of second messenger pathways. In these studies we will investigate the phenotypic and biochemical properties of cells expressing variant FAK proteins and attempt to correlate known defects in FAK activity with alterations in cellular metabolism. Finally, we will characterize the function interactions between FAK and pp60src in src transformed cells and extend this paradigm to the analysis of possible interactions of FAK and pp60src or other src family kinases in normal cells. These experiments seek to delineate the role of pp60src in the structural perturbation of focal adhesions in transformed cells and explore the possibility that FAK regulates or is regulated by src family kinases in normal cells.

DESCRIPTION: (Applicant's Description) The Cellular Imaging Core (Core C) will provide instrumentation and data analysis capability to support the study of intracellular signaling in single cells. This Core will allow us to create and staff a facility that brings cutting edge technology to the analysis of cell structure and cell movement and migration. Understanding signaling in "time and space" requires the development of technologies to visualize signal transduction events in single cells and furthermore to visualize signals within defined compartments of such cells. Thus the Cell Imaging Core is key to the success of each of the projects.

The long term goal of the proposed research is to understand how signal transduction cascades initiated by extracellular matrix-integrin interactions regulate cell adhesion, cell motility, cell growth and differentiation. Our goal is to understand how such signaling pathways are organized (wired) in normal cells and to ultimately define the cellular alterations (both genetic and environmental) that lead to abnormal adhesion signaling in cancer cells. Studies over the past five years have identified two major classes of signaling molecules that participate in and regulate adhesion signaling, protein tyrosine kinases and members of the family of small GTPases. How these two classes of regulatory proteins function to organize the complex signaling required for cell adhesion and movement is a central theme of this proposal. The proposed studies focus specifically on the role of focal adhesion kinase in mediating signals from the extracellular matrix through the beta- integrin receptors. The three aims emphasize the identification and characterization of molecules that directly interact with FAK and link both upstream and downstream signaling components; defining the role of FAK and interacting partners in mediating signals that regulate cell motility and growth; and finally studying how cells utilize a potentially novel mechanism to regulate adhesion signaling during development. The specific aims are: 1) Using our base of knowledge about the structural organization of the domains of FAK and the related protein tyrosine kinase PYK2, identify new structural and functional linkages by defining new binding/interacting partners for FAK; 2) examine the functional role of FAK in the regulation of cell migration, focusing initially on fibronectin induced motility. We will also explore the role of FAK in the regulation of cell motility using FAK null fibroblasts derived from mice containing a conditional deletion of FAK (Cre-mediate deletion of a FAK exon flanked by lox P sites); 3) explore the possible in vivo regulation of adhesion signaling by the cell/tissue type-specific expression of the C-terminal domain of FAK, FRNK FAK-related Non-Kinase) and examine the consequences of knocking out FRNK on the course and extent of normal development of the mouse.

DESCRIPTION (provided by applicant): Research supported by this grant over the past two decades, has focused on using the oncogenic protein tyrosine kinase, Src, as a molecular guide to identify tyrosine phosphorylated proteins that play key roles in cellular signaling. Characterization of so-called "Src targets" has led to the identification of a number of cellular proteins, such as Focal Adhesion Kinase (FAK), p130Cas and paxillin, proteins that function within the context of specific actin-based cytoskeletal structures, e.g., focal adhesions, as well as Cortactin, a protein that localizes predominantly to cortical actin, a site of dynamic actin remodeling. The long range goal of the studies outlined in this proposal is to understand how proteins such as Cortactin, function in the context of the cortical actin cytoskeleton to integrate cell surface receptor mediated cell signaling in a "temporal and spacial" context. Information garnered over the past granting period shows that the Src-substrate Cortactin is a multifunctional adapter protein that interacts on the one hand with the "cortical actin network" present in filopodia and lamellipodia by binding the Arp2/3 complex and links via its carboxyl-terminal SH3 domain to a number of functionally interesting and potentially important signaling and scaffolding proteins. This revised application again outlines four specific aims to address the function of Cortactin: In Aim 1 we will use molecular genetic, cellular and biochemical approaches to study the factors that regulate the interaction of Cortactin with the Arp2/3 complex of proteins. In Aim 2, we will extend and further develop our studies aimed at identifying and characterizing proteins that interact with the Cortactin SH3 domain. We will continue to characterize proteins that function as binding partners for Cortactin, including two recently identified SH3 effectors, WIP, Wasp Interacting Protein, a regulator of Arp2/3-dependent actin polymerization and dynamin, a GTPase implicated in the regulation of endocytic and secretory pathways. We will also extend and further develop studies to identify possible interactions of cellular proteins with the "helical-proline-ser-rich" region of Cortactin, here-to-fore unstudied interaction domain. In Aim 3 we will examine how tyrosine phosphorylation influences the interactions of Cortactin with known cellular binding partners. Finally in Aim 4, we will examine whether expression of full length Cortactin or predicted dominant inhibitory forms of Cortactin function to alter the dynamics of EGF-induced lamellipodia formation or cell migration. We will also determine whether Cortactin or Cortactin variants influence the dynamics of growth factor receptor signaling to downstream effectors, with particular attention to Ras signaling and EGF receptor internalization. Finally we propose to generate mice bearing a genetically altered allele of Cortactin, allowing for Cre-mediated excision and replacement of the Cortactin SH3 domain. Such mice, bearing a mutated form of Cortactin will allow one to directly test the role of Cortactin in mediating specific cellular functions in a cell type specific fashion.

DESCRIPTION (provided by applicant): The five-year survival for patients with pancreatic cancer is less than 5%. Several novel therapies have been shown to be highly effective in mouse models of pancreatic cancer;however, in human clinical trials these agents have failed. There is a compelling need for preclinical models of pancreatic cancer that more accurately reflect the process of disease progression in patients. The objective of this proposal is to develop an orthotopic mouse model of pancreatic cancer using primary human tumors implanted into the pancreas of immune compromised mice. A key component of this model will be the genetic and molecular characterization of tumors and correlation of this with the biologic behavior of the tumors (e.g. grade, invasiveness). The orthotopic model is based on studies described in the literature and previous work of the PI. The establishment of a robust and well characterized orthotopic model will facilitate preclinical testing on an individualized basis of therapeutic agents that target defined signaling pathways important for pancreatic cancer progression and metastasis. A central hypothesis driving the development of the model is that orthotopic implantation of primary human xenografts into the pancreas will more closely recapitulate the growth environment of human pancreatic cancers. Thus, the molecular and cellular analysis of the orthotopically implanted tumors should more accurately reflect the biologic behavior of individual patient tumors, allowing the assessment of the repertoire of cell surface receptor expression/activation and cell signaling pathways activated in each tumor. The Experimental Plan details the following two specific aims: 1) To collect human pancreatic cancer specimens then propagate tumors orthotopically in mice and evaluate growth kinetics, tumor invasion, and metastasis. 2) To perform molecular characterization of human and xenografted tumors and correlate the molecular signaling profile of each tumor with its growth, invasive, and metastatic behavior. While previous studies have described the transplantation of pancreatic tumors subcutaneously and orthotopically, the model described herein will be the first attempt to correlate the clinical properties of pancreatic tumors with the molecular and cellular properties of the tumors propagated orthotopically in the pancreas. In addition to providing fundamental information about the molecular and cellular properties of primary pancreatic cancers, development of this model will be the next step toward arriving at a personalized approach to therapy for pancreatic cancer. PUBLIC HEALTH RELEVANCE: Pancreatic cancer is the fourth leading cause of cancer deaths in the United States and has the shortest survival time of any cancer. Our goal is to develop a novel approach to therapy for pancreatic cancer, with treatment individualized for each patient depending on the best target for therapy of their specific tumor. We plan to achieve this by developing a mouse model of pancreatic cancer in which portions of patients'tumors are grown in the mouse pancreas, and then assessed for their molecular characteristics and response to treatment.