Isa Hussaini - US grants

LP is an endocytic receptor involved in the trafficking of a variety of proteins/protein complexes that have physiologic relevance in the CNS including PNII, lipoprotein metabolites and activated a2M. Receptor associated protein (RAP) copurifies with LRP and blocks cell surface binding of LRP ligands. Regulation of LRP expression might have important biological roles in the nervous system. LRP is developmentally regulated in neurons. In glial cells, LRP appears to be upregulated in certain reactive states and following neoplastic transformation. The hypotheses of this proposal are that: 1) LP and RAP expression in the CNS are dynamically regulated and differentially regulated in astroglial and neuronal cell types and 2) that LRP plays a role in the control of astroglial migration/invasion. The 3 aims are to 1) demonstrate that EGFR ligands and LPS regulated the expression of LRP and RAP in neural cells 2) determine the effect of EGFR ligands and LPS on LRP promoter activity and define the transcription factors responsible for cell type-specific LRP expression in the CNS, and 3) demonstrate the role of LRP expression on neoplastic astrocyte migration/invasion.

DESCRIPTION (provided by applicant): The prognosis for patients with malignant astroglial tumors is poor. The capacity of astrocytomas both to invade adjacent and to migrate into distant brain sites precludes curative surgical resection; and little progress has been made in designing adjuvant therapies that significantly affect long-term survival. In order to formulate more novel therapeutic strategies, it is essential to have a better understanding of how the invasive growth of these tumors is controlled. Recent data from our laboratory using two human glioblastoma cell lines with distinctive growth patterns demonstrate a correlation between expression of protein kinase C-eta (PKC-eta) and a PMA-induced increase in proliferation and decrease in migration. Over-expression of the enzyme in PKC-eta-deficient U-1242 MG cells converted the response to phorbol ester from growth inhibition to proliferation. PKC-eta antisense oligonucleotide and cDNA construct counteracted the response to PMA, suggesting that PKC-eta may be involved in the mitogenic response. This proposal is focused on understanding differences in PKC-eta activation and expression between malignant vs. non-neoplastic astrocytes and on the role of PKC-eta in signaling pathways that control cell proliferation and apoptosis. The regulation of cell proliferation and invasive cell growth in malignant astrocytic tumors is undoubtedly complex; however, the PKC-eta regulation of these processes may be exploited to provide an experimental system in which proliferative and apoptotic phenotypes may be selectively induced and studied. If non-neoplastic and malignant astrocytes have differential regulation of proliferation by specific PKC-isoforms, and if the biologic behavior of the cultured cells can be extrapolated to our animal model, pharmacological regulation of PKC-eta in combination with drugs targeting cellular migration, may present a new therapeutic paradigm for these aggressive brain tumors. I hypothesize that: Expression and activation of PKC-eta are critical in regulating proliferation and apoptosis in astrocytic tumors. The first goal (Aims I &2) of this grant is to determine whether expression and activation of PKC-eta are controlled differently in astrocytic tumor cells and non-neoplastic astrocytes. The second goal (Aim 3) is to (a) determine whether over-expression or deficiency of PKC-eta affects astrocytic tumor growth and apoptosis, (b) identify signaling pathways downstream of PKC-eta mediating these responses, and finally (c) test whether the cell culture data model the in vivo tumor biology. This will be tested in an animal astrocytic tumor model. Understanding the roles of specific PKC isozymes in growth and apoptosis of astrocytic tumors will provide information on possible specific targets for therapeutic intervention.

[unreadable] DESCRIPTION (provided by applicant): The majority of the intracranial tumors are diffuse-type astrocytomas. These gliomas generally have the common biologic features of 1) micro-infiltrative growth into surrounding brain, and 2) a significant potential for malignant progression to occur over time (i.e. in the residual, infiltrating tumor). The first accounts for the failure of surgery alone and inevitable tumor recurrence, even with gross total resection of the tumor mass. The second accounts for the high rate of increased biologic malignancy in the recurrent tumor. These collectively account for the lack of progress in accurate diagnostic grading and therapy for this class of tumors. The paucity of therapeutic innovations in neuro-oncology reflects the limited understanding of how the migration and invasion of these tumors are controlled, so that the invading tumor cells can be more specifically targeted. Low density lipoprotein receptor-related protein (LRP) is an endocytic receptor involved in the trafficking of a variety of protein/protein complexes that have pathophysiologic relevance in the central nervous system, including urokinase (uPA) and its receptor (uPAR), lipoprotein metabolites, and activated alpha2-macroglobulin. Although the function of LRP in the CNS is not clear, there are abundant data in the literature to indicate that some of its ligands may be involved in key pathophysiologic processes. A variety of cytokines and growth factors, including epidermal growth factor receptor (EGFR) ligands, colony stimulating factor-1 and interferon-gamma, regulate the expression of LRP in both peripheral and central nervous systems. Therefore, changes in LRP expression can alter the levels of its ligands and exert significant biologic effects during cellular responses in the nervous system. Astroglial cells in the human brain express LRP and the level of the receptor is altered in certain reactive states and following neoplastic transformation. Based on our preliminary results in this grant and published data, LRP serves a role as an important endocytic receptor that regulates the level of uPA and its receptor (uPAR), known modulators of migration and invasion, around the microenvironment of astrocytic tumors. We hypothesize that LRP expression regulates the migration and invasion of astrocytic tumor cells. The first goal (Aims 1&2) of this grant is to determine whether the regulation of LRP expression differ between neoplastic (primary astrocytomas; glioblastoma cell lines) and non-neoplastic astrocytes and assess how EGFR or PKC-eta activation alters the expression and endocytic function of LRP. The second goal (Aim 3) is to determine whether overexpression or deficiency of LRP or its ligands (uPA/uPAR) affects astrocytic tumor migration and invasion in vitro and in vivo. Understanding the role of LRP in the migration and invasion astrocytic tumors will provide information on possible specific targets for therapeutic intervention. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The prognosis for patients with malignant astrocytic tumors is poor. The capacity of astrocytomas both to invade adjacent brain sites and to migrate into distant ones precludes curative surgical resection;and little progress has been made in designing adjuvant therapies that significantly affect long-term survival. In order to formulate more novel therapeutic strategies, it is essential to have a better understanding of how the invasive growth of these tumors is controlled. Our animal model and in vitro results suggest that even though PKC-eta expression increases mitogenic response, the glioblastoma cells are less invasive. Our working hypothesis is: Expression and activation of PKC-eta decrease astrocytic tumor invasion. Three aims are proposed to test the hypothesis (I) To determine the factors controlling the expression of PKC-eta in astrocytic tumor cells (II) To determine the role and mechanism of PKC-eta-mediated decrease in glioblastoma invasion in vitro and (III) To assess the role of PKC-eta expression on GBM invasive growth in a xenograft GBM NOD- SCID mouse model using immunohistochemistry, tissue slice migration and magnetic resonance imaging (MRI). PUBLIC HEALTH RELEVANCE: The malignant form of astrocytoma known as glioblastoma multiforme is most commonly diagnosed primary brain tumor. This tumor is resistant to chemotherapy and radiation. There are multiple factors and pathways that are involved in the formation and progression of glioblastoma multiforme. This research project has identified a novel PKC isozyme (PKC-eta) as a molecular switch that when expressed in GBM, cells become highly proliferative and less invasive. In contrast, when this kinase is absent the GBM cells become highly invasive. Furthermore, we have identified a putative downstream target (MMP-9) of PKC-eta and a transcription factor (NF-kB) that may explain one of the many mechanisms for controlling GBM invasion both in vitro and in vivo. Understanding the functional roles of these genes in GBM invasion may provide a paradigm whereby this deadly tumor may be managed.