Marsha Rich Rosner - US grants

Tumor promoters accelerate the process of cellular transformation through alteration of normal cellular growth and differentiation processes. One approach that has yielded important information regarding the regulation of growth factor receptors and the mechanisms by which tumor promoters act is to study the interaction of tumor promoters with receptor systems. Thapsigargin is a protein kinase C-independent tumor promoter that has recently been shown to alter epidermal growth factor (EGF) receptor binding and kinase activity. Evidence suggests that the response of the receptor is due to activation by thapsigargin of a kinase that phosphorylates select sites on the EGF receptor that differ from those phosphorylated by protein kinase C. We now propose to identify these sites and to directly test their role in the action of thapsigargin through site-directed mutagenesis studies of the EGF receptor. We also plan to investigate the mechanism by which thapsigargin stimulates kinase activation. These studies provide a new approach to understanding the regulation of cellular receptors by exogenous agents, and will lead to the elucidation of new signal transduction pathways in the cell.

It is the goal of this study to understand the regulation and expression of protein kinase C, a major receptor for tumor promoters which has been implicated in the process of hormone-mediated cellular mitogenesis as well as tumor promotion. Having demonstrated that the epidermal growth factor receptor is a biological target of protein kinase C action, our initial aim is to use this system following treatment with tumor promoters to determine the number and nature of the different activation states of C kinase. Once the level of active enzyme required to induce a biological response is determined, we will explore the role of C kinase in the action of different growth enhancers on nontransformed cells. The differences in protein kinase C activity in transformed relative to nontransformed cells will be analyzed, as well as the causes of C kinase inactivation. Finally, we plan to prepare antibody directed against protein kinase C as an aid in achieving these goals. An understanding of the factors which regulate the activation of protein kinase C should provide valuable information regarding the mechanisms of mitogenic stimulation, hormonal regulation, and the onset of tumor promotion.

ABSTRACT NARRATIVE Description: This proposal requests funds for the travel of nine U.S. scientists to an International Conference on the Structure and Function of Biomembranes to be held at the Bose Institute, Calcutta, India, November 30-December 2, 1989. The U.S. principal investigator, Dr. Marsha Rosner of the University of Chicago is cooperating with the Indian converner of the conference, Dr. P. Chakrabarti of the Bose Institute. The conference is decribed as a one-of-a-kind international conference to address fundamental issues of membrane structure and function. The organizing committee consists of a noted scientist from the following countries: United States, United Kingdom, West Germany, Belgium and India. Scope: The proposed conference will provide a unique opportunity for American scientists working in membrane-related areas to establish collaborations and relationships with researchers from all participating countries. Membrane research in the beginning covered a broad range of subjects, but soon began to specialize in narrowly focused fields of research. This specialization is now seen as a disadvantage in loss of perspective and knowledge that other disciplines may provide membrane research. This cross-disciplinary conference is a means to open-up and advance new ideas and information in the field. This proposal fulfills the criteria of the U.S. - India Cooperative Science Program regarding the support of international conferences and workshops dedicated to the exchange of information and to expand scientific cooperation.

Although maintaining the integrity of the central nervous system (CNS) is of extreme importance to human health and function, relatively little is known about the signaling processes involved in the proliferation, differentiation and survival of CNS neurons. Two growth factors, epidermal growth factor (EGF) and fibroblast-derived growth factor (FGF), have been implicated as mitogens, differentiating agents, and neurotrophic agents for neuronal stem cells and neurons in a number of CNS regions, including the hippocampus. The hippocampal-septal axis plays a key role in learning and memory, is involved in many neuropathies including Alzheimer's disease, epilepsy and stroke, and has a relative structural simplicity which has proven useful in studies of development and plasticity. In order to investigate the mechanism by which growth factors influence hippocampal development and function, we have generated neuronal cell lines of hippocampal lineage that proliferate in response to EGF and differentiate into a nonproliferating, neuronal phenotype in response to bFGF. In the present proposal, we will focus on the mechanism by which growth factors differentiate neuronal cells. We propose to use these cell lines to define major signalling intermediates in the bFGF signal transduction pathways and test the hypothesis that these intermediates are responsible for the actions of bFGF as a differentiating agent/ Specifically, we plan to identify activated intermediates in the FGF signal transduction pathway, determine the role of activated intermediates in the FGF signaling pathway, and identify and characterize genes that may code for novel intermediates in the signaling pathways leading to differentiation of H19-7 cells. The results of these studies will define key signaling intermediates that influence neuronal differentiation and increase our understanding of the processes that lead to neoplastic transformation and neurodegenerative disease involving neuronal differentiation and survival.

Increased airway smooth muscle mass is thought to contribute to the airway hyperresponsiveness observed in patients with asthma. The potential importance of abnormal airway smooth muscle cell proliferation highlights the need for an understanding of the early events involved in airway smooth muscle mitogenesis. To achieve this overall goal, we propose the following Specific Aims: SPECIFIC AIM 1: Determine the precise role of cyclin D(1) in bovine tracheal myocyte G(1) traversal. We will assess cyclin D(1) expression and cyclin D(1)-dependent kinase activity following mitogenic stimulation by immunoblotting and measurement of retinoblastoma protein phosphorylation. The requirement and sufficiency of cyclin D(1) for bovine tracheal myocyte cell cycle progression will be assessed by stable transfection of cells with either cyclin D(1) antisense or sense cDNA using the inducible TET repressor system, followed by flow cytometry and cell counting. The effects of TGFbeta and forskolin, an activator of adenylate cyclase, on cyclin D(1) synthesis, associated kinase activity and cdk inhibitor expression will be determined. SPECIFIC AIM 2: Identify downstream targets of MAPK in bovine tracheal myocytes. We will assess cyclin D(1) promoter transcriptional activity in cells transiently- transfected with the cyclin D(1) promoter, subcloned into a luciferase reporter. Luciferase activity will be measured after stimulation with mitogens and non-mitogens, as well as following co-transfection with plasmids encoding dominant-negative or constitutively-active forms of MEK- 1, a dual-function kinase required and sufficient for MAPK activation in these cells. The cis-acting DNA sequences required for MAPK-induced cyclin D(1) promoter activity will be assessed with a series of cyclin D(1) 5' flanking region deletion mutants. We will identify the precise nuclear transcription factor(s) involved in this transcriptional regulation by performing gel mobility shift assays, DNase footprinting and Southwestern blotting. SPECIFIC AIM 3: Identify alternative, Raf-1- independent upstream activation pathways of MAPK in bovine tracheal myocytes. Kinase activity for MEK-1 will be isolated from forskolin and PDGF-treated cells using a Mono-Q column and assessed by in vitro phosphorylation assay. Fractions with kinase activity will be probed with antibodies against MEK kinase (MEKK), Raf-1, A-Raf, B-Raf and Mos. If detected by immunoblotting, the role of the MEK activator(s) will be confirmed by sequential immunoprecipitation, followed by re-testing for MEK kinase activity. If the MEK activator is novel, it will be purified by column chromatography, visualized by active-site labeling, microsequenced and cloned. This work may shed light on parallel mechanisms that may operate in asthma, and lead to therapeutic interventions.

The mitogen-activated protein kinases (MAPKs) are a superfamily of highly homologous proline-directed serine/threonine kinases that participate in the transduction of growth and differentiation-promoting signals, as well as stress responses to the cell nucleus. The presence of at least six MAP kinases in yeast suggests that there are likely to more in mammals. In order to detect additional MAP kinases, we screened a rat brain library using degenerate polymerase chain reaction (PCR) and identified a novel MAPK termed ERK7 that encodes a 61 kD, 546 amino- acid long protein ERK7 contains the Thr-Glu-Tyr (TEY) activation motif characteristics of other ERKs, but has a number of properties that are unique. ERK7 has a discrete C-terminal domain that contains SH3 binding motifs. Unlike other ERKs, ERK7 has significant constitutive kinase activity, and this activity is dependent upon the C-terminal domain. Furthermore, ERK7's C-terminal domain rather than its kinase activity is required for nuclear localization and its function as an inhibitor of DNA synthesis. Finally, ERK7 is the first MAP kinase that has been found to specifically associate with a protein that activates chloride ion transport, CLIC3, and the C-terminal domain is sufficient for CLIC3 binding. The identification of a MAPK with C-terminal SH3-binding domains and the importance of the C-terminus in ERK7 function suggests that ERK7 represents a subfamily of MAPKs with adaptor domains that contribute to signaling specificity in growth and development. In this application, we propose to further characterize the regulation and function of this novel kinase. Specifically, we plan to use molecular and cellular approaches to 1) Determine the mechanism by which ERK7 is activated; 2) Identify key functional domains, binding partners and potential substrates of ERK7; and 3) Investigate the function of ERK7 in cell and tissues. The results of these studies will test the hypothesis that ERK7, like other ERKs, play a key role in the regulation of cell growth and tumor progression, and will increase our understanding of this new member of the MAPK family.

DESCRIPTION (provided by applicant): Squamous cell carcinomas of the head and neck (SCCHN) generally overexpress epidermal growth factor receptors (EGFR) that have been linked to poor prognosis, treatment failure, and shortened survival. Protein kinase C enzymes (PKCs) have also been implicated in growth control, particularly as mediators of EGF signaling. Recently, we have demonstrated that the atypical protein kinase C isoform, PKC zeta, is necessary for EGF induced MAPK activation in head and neck cancer cell lines. PKC zeta has also been implicated in activation of p70 S6 kinase and the survival factor Nuclear Factor kappa B (NF-kappaB), and we have shown that inhibition of PKC zeta promotes apoptosis in head and neck tumor cells. Finally, we have shown that general inhibitors of classic and novel PKCs are cytotoxic to SCCHNs in vitro and in vivo, but the specific PKC isoforms responsible have not been identified. The present proposal seeks to identify the PKC isoforms that regulate signaling pathways that lead to SCCHN growth and survival, and to determine the potential efficacy of PKC inhibitors as therapeutic agents for the treatment of SCCHN. Specifically, we plan to: 1) Determine the nature of PKC expression in human tumor and normal tissues collected during clinical trials in squamous cell carcinoma of the head and neck; 2) Characterize the role of PKC isozymes in EGF and serum stimulation of head and neck tumor cells; 3) Test the effect of PKC inhibitors on apoptosis of head and neck tumor cells either alone or in combination with radiation, EGFR inhibitors, or TNF-alpha; and 4) Determine the effect of PKC targeted agents on head and neck cancer in vivo.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] Activation of growth signaling cascades by epidermal growth factor (EGF) and other mitogens is a key step in cell growth and development as well as the generation of neural and other epithelial-derived tumors. Among the main mediators of signals transmitted by tyrosine kinase receptors such as the EGF receptor are the mitogenactivated protein kinases (MAPKs), a superfamily of highly homologous proline-directed serine/threonine kinases that participate in the transduction of growth and differentiation-promoting signals. The Ras/Raf kinase cascade that leads to activation of ERK1 and ERK2, two members of the ERK subfamily of MAPKs, is required for cell growth and thus is an important target of therapeutic agents for tumor treatment. Recent studies from our laboratory have revealed a new mechanism for the regulation of Raf-1 by growth factors. Under nonstimulatory conditions, Raf-1 can form a complex with a Raf Kinase Inhibitory Protein (RKIP/PEBP). An evolutionarily conserved protein that is present from bacteria to man, RKIP prevents Raf- 1 from downstream signaling. We have shown that different growth factors activate specific members of the protein kinase C (PKC) family that, in turn, cause release of RKIP, enabling activation of Raf and the MAPK signaling cascade. More recent results have implicated RKIP in mitotic progression. We therefore hypothesize that RKIP is a key regulator of mitogenesis. The goal of this proposal is to characterize further the physiological functions of RKIP, elucidate the mechanisms by which RKIP functions, and define the structural interactions that underlie these mechanisms. Specifically, we plan to (1) Determine the role(s) of RKIP at different stages of the cell cycle in regulating cell growth; (2) Determine the mechanism(s) by which RKIP regulates MAP kinase signaling and mitotic progression; and (3) Identify the key functional domains in RKIP. The studies proposed here will increase our understanding of the regulation of these key events in cell proliferation, and should lead to the development of new reagents that can inhibit growth of neural and other epithelial-derived tumors. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Dysregulation of growth signaling cascades is a key step in the initiation and malignant progression of prostate cancers. Among the main mediators of signals in proliferative cells are the mitogen-activated protein kinases (MAPKs), a super-family of highly homologous proline-directed serine/threonine kinases that participate in the transduction of growth and differentiation-promoting signals. The Ras/Raf kinase cascade that leads to activation of ERK1 and ERK2, two members of the ERK subfamily of MAPKs, is required for normal cell growth as well as neoplastic processes. Activation of this signaling cascade is correlated with prostate cancer progression and androgen-independence and thus is an important therapeutic target. One of the modulators of Raf kinase is Raf Kinase Inhibitory Protein (RKIP). Recent studies from our laboratory have shown that RKIP regulates Raf activation via interaction at S153;phosphorylation of S153 by activators of protein kinase C release RKIP, enabling subsequent activation of Raf. We have also shown that RKIP regulates mitotic progression in a number of cell types. An exciting new biologic function for RKIP in the suppression of prostate cancer metastasis has recently been reported. Specifically, immunohistochemical analysis of clinical tissue samples revealed expression of RKIP in primary prostate cancer but not in metastatic lesions. Functional in vivo studies showed that ectopic expression of RKIP suppressed invasion in vitro and metastasis in vivo in the well-characterized metastatic C42 human prostate cancer cell model. Conversely, decreasing the level of RKIP in nonmetastatic LNCaP cells promoted their invasive ability in vitro. Taken together, these studies implicate RKIP as a metastasis suppressor protein. We anticipate that identification of the mechanism by which RKIP signals to suppress metastasis will provide information necessary to understanding the molecular underpinnings of prostate cancer metastasis. The goal of this proposal is to relate RKIP structure to its function as a metastasis suppressor and then use this information to develop therapeutic reagents that modulate or mimic RKIP biologic function. Specifically, we plan to: 1) Identify the targets of RKIP action in prostate tumor cells;and 2) Generate inhibitors or potentiators of Raf-1 or RKIP. The reagents developed in these studies could be used in combination with other tumor therapies to suppress specific steps in the metastatic cascade.

DESCRIPTION (provided by applicant): Women with triple-negative breast cancer (TNBC) have the shortest survival and highest relapse rates; however, there are no known targeted therapies. Metastasis, the cause of most cancer-related deaths, is dependent upon specific molecular interactions between tumor cells and their microenvironment. We recently conducted a study to characterize these tumor-stromal interactions in a TNBC xenograft model. To identify drivers of metastasis, we compared gene expression in an invasive tumor, and a genetically matched noninvasive tumor that stably expressed the metastasis suppressor Raf Kinase Inhibitory Protein (RKIP). Our studies revealed a striking loss of macrophage infiltration in noninvasive (RKIP+) tumors when compared to invasive tumors. Preliminary results suggest that RKIP inhibits expression of the macrophage chemokine, CCL5, in tumor cells and its receptor, CCR5, in the stroma. CCL5 overexpression in noninvasive (RKIP+) tumors partially rescued macrophage infiltration suggesting that CCL5 is sufficient to initiate metastasis. The CCL5-CCR5 axis has been implicated in BLBC metastasis, but its mechanisms of action and the role of macrophages are not known. We hypothesize that the chemokine CCL5 mediates interaction between TNBC cells and tumor-associated macrophages to drive invasion and metastasis AND that blocking CCL5 activity combined with targeted EGFR therapy in humanized mice will lead to reduced tumor and metastatic burden. We propose to elucidate the nature and role of these interactions. Specifically, we will: 1) Characterize CCL5-mediated crosstalk between TNBCs and TAMs; 2) Investigate the role of TAMs and Ccl5 in a TNBC mouse model; and 3) Investigate the mechanism by which RKIP regulates CCL5 expression and its potential therapeutic application. Our studies could both inform clinicians about risk factors leading to triple-negative breast cancer as well as lead to potential therapeutic targets.

The overall goal of this proposal is to understand the mechanism by which Raf Kinase Inhibitory Protein (RKIP) utilizes phosphorylation to switch between three functional states and regulates the cellular kinome. RKIP is the prototypical member of the Phosphatidylethanolamine Binding Protein (PEBP) family, which comprises more than 400 proteins that are evolutionarily conserved both in sequence and structure, spanning from bacteria to humans. In spite of its relatively small size (185 residues), RKIP plays a dual role as a suppressor of metastatic cancer and as a regulator of cardiac function. Importantly, its dysregulation can lead to disease states. In the kinase signaling cascades, RKIP functions both as sensor and effector. As an effector, RKIP modulates allosterically the activity of different kinases, depending on its phosphorylation state. Specifically, RKIP regulates key mammalian signaling cascades, including MAP kinase (MAPK) and G protein- coupled receptors (GPCRs). Phosphorylation by Protein Kinase C (PKC) switches RKIP function from inhibiting Raf/MAPK signaling to inhibiting G-protein-coupled receptor kinase (GRK2), thereby up-regulating the ß-adrenergic receptor (ß-AR) and its downstream target Protein Kinase A (PKA). While solid biological data are available, little is known about the molecular mechanisms. We now propose that RKIP functions via a novel regulatory mechanism, where phosphorylation of RKIP acts on an existing salt bridge and triggers an allosteric switch of function. As a sensor, RKIP responds to changes in the MAPK and PKA signal transduction pathways through an unknown mechanism. It has been hypothesized that RKIP would function as a simple two-state system, with RKIP binding to either Raf (RKIPRaf) or GRK2 (RKIPGRK2). However, compelling data from our lab indicate that RKIP adopts three discrete functional and conformational states. The additional, high energy intermediate state (RKIPKin) is the one responsible for the interaction with the kinase cascades. Based on our data, we propose a novel positive feedback loop, where kinases that are downstream of RKIP targets bind and phosphorylate the RKIPKin state. Phosphorylated RKIPKin (pRKIPKin) promotes further phosphorylation of RKIP triggering the phospho-switch. In this proposal, we will characterize the structures and functions of RKIP in its allosteric states, combining our expertise in biophysics and signal transduction. We will accomplish the following specific Aims: 1) Characterize the phospho-switch and the RKIPGRK2 state; 2) Characterize the nature and function of the allosteric switch to a high energy state; and 3) Test the effects of allosteric states defined by biophysical studies on RKIP function in cancer. Although the immediate goal is to understand the signaling role of RKIP, the concepts developed in this grant application will help understand a novel relationship between phosphorylation and allostery that will be of general importance in cell signaling and communication.