Danny Dhanasekaran - US grants

G-proteins are involved in the signal transduction processes ranging from sensory perception to cellular growth control mechanism. The impairment of G-protein function has been implicated in several pathological conditions such as pseudohypothyroidism, cardiac dysfunction, and oncogenesis. Constitutively activated G-protein pathways have been demonstrated to cause cellular transformation. The precise mechanism by which these mutations in G-proteins disrupt normal cell growth has not been fully understood. In addition, the growing list of G-protein of subunits has left several members of this class of signal-transducers without defined receptors or effectors. G-alpha12 and G-alpha13 are the members of G 12 family of G proteins. GTPase deficient mutants of G-alpha12 and G-alpha13 readily transform the fibroblast cell lines. Previously we have shown that these alpha-subunits activate Na+/H+ exchangers and unique set of primary response genes. Our recent studies indicate that these alpha-subunits also activate the newly identified Jun N-terminal Kinase (JNK) family of Mitogen Activated Protein Kinases (MAP kinases). Specifically this proposal will focus on identifying the signaling components regulated by G-alpha12 and G-alpha13 in relation to their oncogenicity of these alpha- subunits. The specific aims of this proposal are 1. To define the role of G-alpha12 and G-alpha13 in activating growth factor-sensitive Na+/H+ exchanger; 2. To analyze the mechanism of G-alpha12 and G-alpha13 activation of specific set of primary response genes; and 3. To analyze the mechanism of G-alpha12 and G-alpha13 activation JNK. The long term objective of this project is to identify the role of G-protein regulated signal transduction pathways in relation to cell growth and development.

Heterotrimeric G proteins, G12 and G13 regulate critical pathways involved in cell growth, differentiation, and apoptosis. The studies outlined here will investigate the mechanism(s) by which the alpha-subunit of G12, Galpha12 regulates distinct signaling pathways in the context of cell proliferation. The following experimental approaches will be taken: I. Analysis of the mechanism of signal coupling between Galpha12 and small GTPases in activating proliferative pathways: Studies from several laboratories including ours have shown the Galpha12- activated JNK plays a critical role in Galpha12-mediated mitogenic pathway. Galpha12 stimulates JNK-signaling modules through the small GTPases Ras, Rac, CDC42, and Rho. The mechanism(s) through which Galpha12 couples to these small GTPases in activating the kinase modules and cell proliferation will be analyzed. II. Analysis of the role of kinase signaling modules in Galpha12- stimulated cell proliferation: The preliminary studies have shown that Galpha12QL potently activates JNK and acutely inhibits p38MAPK through the differential regulation of the different MAP kinase kinases. The present study will be focused on 1) identifying molecular basis for the differential regulation of these kinases by Galpha12QL and 2) defining the role of such differential regulation in Galpha12QL-stimulated mitogenic pathway. III. Analysis of the role of small GTPases and kinases in Galpha12-stimulated COX-2 pathway: Galpha12QL stimulates the expression of several unique primary and secondary response genes including Egr-1, Jun B, and COX-2. Since the stimulation of COX- 2 can lead to the development of an autocrine loop, the mechanism through which Galpha12QL stimulates the expression of COX-2 and its role in Galpha12-mediated cell proliferation will be analyzed. The studies presented here will identify the crucial signaling pathway(s) involved in Galpha12-mediated cell proliferation and transformation. In light of the proposed causative role of Galpha12 in soft-tissue sarcoma, these studies are anticipated to provide valuable clues to the etiology of specific types of soft- tissue sarcomas such as malignant fibrous histiocytoma. The long-term goal of this project is to elucidate the mechanisms by which G proteins integrate signaling from different pathways in the regulation of cell proliferation.

DESCRIPTION (provided by applicant): Metastasis involves the expression of different proteins involved in cell motility along with homing receptors, their ligands, and extracellular matrix degrading proteases. Recent studies have identified that the signaling pathways regulated by lysophosphatidic acid (LPA) play a critical role in ovarian cell growth and metastasis. Based on our preliminary findings that G?13 (which can be activated by LPA) and Hax-1 interaction is required for cell motility, we hypothesize here that G?13-HAX-1-cortactin interaction is critically involved in the metastasis of LPA-responsive ovarian cancer cells. This hypothesis will be tested under the following specific aims: Aim 1: Characterization of G?13-Hax-1 interaction. Hax-1 interaction sites with G?13 and cortactin will be defined by site-directed mutagenesis approach;Aim 2: Analysis of G?13-Hax-1-cortactin complex. The interrelationship of Hax-1, G?13, Cortactin, and Rac in forming the quadnary complex and the role of Hax-1 and G?13 in cortactin-phosphorylation will be defined;Aim 3: Analysis of other Hax-1 interacting signaling components. The target pathways regulated by G?13-Hax-1 complex will be identified by the analysis for specific GEFs and regulation of uPA and/or MMP2;Aim 4: Effect of Hax-1 on the oncogenic activity of G?13: We will investigate whether G?13-Hax-1 association is involved in neoplastic growth of ovarian cancer cells;and Aim 5: Role of G?13-Hax in tumor cell migration and metastasis. The role of Hax-1 or G?13 on the migration and invasive potentials of ovarian cancer cells will be analyzed using the novel mouse model system developed by Dr. Sandra Orsulic (Co-P.I) at Massachusetts General Hospital, Boston. We hope that the identification of a signaling locus involved in tumor cell motility, as proposed here, will define newer targets for therapeutic intervention.

[unreadable] DESCRIPTION (provided by applicant): This R21 application (in response to PA-06-303, titled "Pilot Studies in Pancreatic Cancer") is based on our recent finding that lysophosphatidic acid (LPA), a simple, bioactive glycerophospholipid that stimulates its cognate G protein coupled receptors (GPCRs) can activate both oncogenic cell growth and cell migration in addition to transactivating c-Met in pancreatic cancer cells. LPA, the ligand for a family of LPA-receptors (LPARs), has emerged as a factor of biological importance in the progression of ovarian as well as pancreatic cancers. Recent studies have shown that LPA can stimulate pancreatic cancer cell migration as well as the transactivation of c-Met, which is known to be critically involved in the motility of many cancer cell lines. However, relatively little is known about underlying mechanisms. In this context, our recent findings that the gep oncogene G mediated transactivation of receptor tyrosine kinases and cell movement are of great significance. Based on these findings, we hypothesize that a specific LPA-receptor stimulates the progression of pancreatic cancer via distinct heterotrimeric G proteins and the downstream small GTPases. This hypothesis will be tested under the following specific aims: Aim-1. Define the LPA-receptor(s) involved in the transactivation of c-Met, migration, and invasion of pancreatic cancer cells. Using siRNA specific to each of these receptors, we will define the receptor that mediates the transactivation of c-Met and invasive migration of pancreatic cancer cell lines using a panel of cancer cell lines consisting of BcPC3, Dan G. MDAPanc-28, and Mia PaCa-2 cells. Aim-2. Define the heterotrimeric G protein that couples LPARs to cellular responses involved in the transactivation of c-Met, migration, and invasion. LPARs have been shown to transmit their messages via the heterotrimeric G proteins defined by the G mutants of the respective LPAR (identified in the previous aim) to the intracellular effectors. In addition to characterizing novel etiological factors involved in the progression of pancreatic cancer, the outcome of these studies is expected to identify novel therapeutic targets for the treatment of the disease. These studies will identify and characterize novel etiological factors involved in the genesis and progression of pancreatic cancer. In addition, the outcome of these studies is expected to identify novel diagnostic, prognostic and therapeutic targets for pancreatic cancer. [unreadable] [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Ovarian cancer is identified as the fifth leading cause of cancer death in the United States with approximately 16,000 deaths every year. Although major advances have been made in understanding the pathology of this disease, 52 % of patients die because of the aggressive growth of these cancers. It has been recently observed that the lipid growth factor lysophosphatidic acid (LPA) promotes ovarian cancer genesis and progression. Our recent studies have shown that LPA-mediated oncogenic signaling involves the gep oncogenes, defined by the activated mutants of G?12 and G?13, that differentially regulate a number of MAP kinases including Jun N-terminal kinase (JNK). These MAPKs, in turn, elicit a multitude of cellular responses required for different physiological stimuli. The signaling complexity and precision of G?12/13 indicates the possible involvement of a scaffolding protein that can modulate specific signaling responses. Recently we have shown that these gep oncogenes, upon stimulation with LPA, interact with a scaffolding protein involved in the activation of JNKs (Kashef et al., 2005). This novel scaffolding protein known as JNK-interacting Leucine- zipper Protein (JLP) potentiates the activation of JNK by G?12/13. In addition, our studies indicate that JLP also tethers ?-PIX, a Rac/CDC42-specific guanine nucleotide exchange factor, to G?12/13 through which the JNK-module can be activated. Based on these results, we hypothesize that G?12/13-JLP interaction is critically involved in G?12/13-mediated activation of diverse cellular responses. In this application, we propose to test this hypothesis under the following specific aims: Aim 1: Characterization of G?13-JLP-?-PIX interaction through site-directed mutagenesis; Aim 2: Analysis of the interrelationship of G?13, JLP, and ?-PIX in forming a signaling complex; Aim 3: Defining the role of JLP on the oncogenic activity of G?12/13 and Aim 4: defining the Role of JLP- G?12/13 signaling in ovarian cancer genesis and progression. In addition to characterizing the etiological factors involved in the progression of ovarian cancer, the outcome of these studies is expected to identify novel therapeutic targets for the treatment of the disease. [unreadable] [unreadable] [unreadable]

The Adiministrative and Mentoring Core will be responsible for all fiscal activities related to the COBRE program, including the placement of all orders and providing financial reports to the module directors and investigators. Dr. Dhanasekaran will direct the Adiministrative and Mentoring Core, which will provide all administrative functions of the COBRE program. He will be assisted by a Business Administrator and a Staff Assistant. The Business Administrator, Mr. Bobby Thomas (25%), will oversee the activities of an Administrative Assistant, Ms. Alisha Booth (100%). Mr. Thomas has been with the OUHSC for 10 years and is very convensant with the fiscal and grant management policies of OUHSC and federal funding agencies including NIH. Mr. Thomas will meet monthly with each investigator to review their accounts and mentor them in the fiscal management of sponsored research grants. Salaries for the PI, administrative staff, mentoring faculty, core directors will be paid through this core module. In addition, this core will administer the fiscal accounting for the proposed pilot projects. Coordinating meetings, travel and activities of the Internal Advisory Committee and the External Advisory Committee will be the responsibility of the Administrative Assistant, Ms. Booth, under the direction of the PI.

DESCRIPTION (provided by applicant): The overall objectives of is to Improve the quality and quantity of cancer research at the University of Oklahoma Hearth Sciences Center (OUHSC), to increase NIH funding in Oklahoma through the mentoring of promising junior investigators (PJIs) and to develop support for the infrastructure for cancer research, this COBRE grant will greatly strengthen cancer research in Oklahoma and enhance the research base, infrastructure, and core capacities of the Cancer Center. The focused and structured mentoring experience that the COBRE grant supports will be a determinative influence helping PJIs achieve independent national funding. Finally, as has been seen on campus in the areas of Vision, Diabetes, Dental Sciences and Microbial Pathogenesis, the CoBRE grant will help accelerate the development of the Cancer Center as it progresses towards a submitting a competitive NCI Cancer Center Support Grant application. The Specific Aims of this CBRE application are: 1. To mentor promising junior investigators (PJIs) In cancer research, enabling them to become independent NIH-funded investigators. Four PJIs have been selected. Each has been assigned a mentor and a co-mentor who are established cancer researchers in Oklahoma with expertise related to the PJI's project. The four PJIs have diverse but related projects that are integrated under the overall theme of Tumor Biology: Resistance to Cancer Therapy and Mitigating Strategies. 2. To establish and develop research infrastructure to support cancer research, optimize the research environment for the PJIs, and form a core resource for the developing Stephenson Oklahoma Cancer Center. Four shared Cores will be established in this COBRE to assist the PJI projects and to serve the cancer research community in Oklahoma. 3. To foster and enhance collaborations between basic scientists and clinicians, and to facilitate translational research directed toward the development of new diagnostics and treatments for cancer.

Abstract ? Overall Component The overall objectives of the proposal are to improve the quality and quantity of cancer research and increase NIH funding at the University of Oklahoma Health Sciences Center (OUHSC), Oklahoma through the mentoring of promising junior Target investigators (Target investigators) and to develop support for the infrastructure for cancer research. Building on the success of the Phase I of this COBRE, the Phase II COBRE grant will support and sustain the infrastructure developed during the previous Phase I funding period in addition to mentoring a new cadre of target junior investigators. This COBRE will continue the process of strengthening cancer research in Oklahoma and enhance the research base, infrastructure, and core capacities of the Cancer Center. The focused and structured mentoring process of this COBRE will have a determinative influence helping TJIs achieve independent national funding. Finally, as has been observed with the Phase I of this COBRE, the Phase II grant will accelerate the development of the Cancer Center as it progresses towards submitting a competitive NCI Cancer Center Support Grant application. The overarching objective of the COBRE in enhancing the cancer research capacity of Oklahoma will be accomplished through the following specific aims: 1) To sustain the successful mentoring of promising junior target junior investigators in cancer research, enabling them to become independent NIH-funded investigators; 2) To support and strengthen the research infrastructure established during the Phase I of this COBRE; and 3) To foster and enhance multidisciplinary collaborative research to facilitate translational research.