Mingyao Liu - US grants

DESCRIPTION (provided by applicant): Among the drugs and agonists that are known to relax the vascular smooth muscle and reduce high blood pressure, beta-adrenergic agonists and nitric oxide-containing compounds are some of the most effective ones. The mechanisms of these drugs are thought to inhibit intracellular free calcium concentration by elevating intracellular cAMP and cGMP, and by activating protein kinases, respectively. However, the molecular targets of cyclic nucleotide-dependent protein kinases are not clear. The molecular basis for the regulation of the vascular smooth muscle by some popular drugs is not well defined. Phospholipase C (PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to generate two important second messengers, diacylglycerol and inositol 1,4,5-triphosphate (InsP3), leading to the activation of protein kinase C and the mobilization of intracellular Ca2+. Detailed characterization of the PLC-B subfamily of proteins will reveal specific receptors, G-proteins, and regulators with which PLC interacts in various signaling pathways, in different developmental and physiological contexts. Thus, PLC enzymes will be used as a "biological probe" for the identification, characterization, and functional dissection of a family of molecular components that collaborates to regulate specific cellular responses in the vascular smooth muscle. Our aims during this grant period are to investigate the regulation of PLC mediated Ca2+ signaling both horizontally, by signaling pathways coupled to cAMP/PKA and cGMP/PKG, and vertically, by interacting with other signaling molecules in the cell. Specifically, we will: 1) Determine the phosphorylation of PLC-B isozymes by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG), respectively. 2) Define the role of protein phosphorylation in G-protein mediated PLC activation and Ca2+ signaling in the vascular smooth muscle cells. 3) Characterize a novel human PLC-B3 interacting protein (hPIP1) in PLC-Ca2+ signaling. Together, we will understand the molecular basis for the modulation of G-protein mediated PLC-Ca2+ signaling in the vascular smooth muscle cells and the regulation of Ca2+ homeostasis in general.

DESCRIPTION (provided by applicant): Receptors coupled to heterotrimeric GTP-binding proteins (G-proteins) are integral membrane proteins involved in the transmission of signals from the extracellular environment to the cytoplasm. A variety of external stimuli, including neurotransmitter, hormones, phospholipids, and growth factors, can activate the G-protein coupled receptors (GPCRs). Therefore, the G-protein coupled receptors represent important specific targets for a variety of human diseases, ranging from the control of blood pressure, allergic response, kidney function, and hormonal disorders, and neurological diseases to human cancers. Most recently, we have identified a novel human prostate-specific G-protein coupled receptor (PSGR). Although human PSGR shares sequence homology with other GPCRs in the seven putative transmembrane domains, the expression of PSGR is highly restricted to the epithelial cells of human prostate gland, not in any other human tissues. PCR and Matched Normal/Tumor Tissue Array study shows significant over-expression of PSGR mRNA in prostate tumor tissues. In this grant proposal, we will test the hypothesis that PSGR is a prostate-specific G-protein coupled receptor and a potential molecular target in human prostate cancers. Two specific aims are proposed in our study: First, we will develop fully human antibodies against human prostate-specific G-protein coupled receptor (PSGR) using a highly complex human pACT2-scFv library. Second, we will determine the potential roles of PSGR as a molecular target in prostate cancers using human PSGR antibodies. We will also investigate the possibility of adding a toxin (or a prodrug) to the antibody and test the hypothesis that the antibody-toxin fusion proteins can directly and specifically bind to and kill the PSGR over-expressing prostate cancer cells. The long-term benefits of the proposed research are the potential of the human anti-PSGR antibodies as agents in the treatment of human prostate cancers and PSGR receptor as a molecular target in human prostate cancer therapy.

DESCRIPTION (provided by applicant): Metastasis is a major cause of death in cancer patients and involves multistep processes including detachment of cancer cells from a primary tumor, invasion of surrounding tissue, spreading through circulation, re-invasion and proliferation in distant organs. KiSS-1 is a human metastasis suppressor gene that has been shown to inhibit metastases of human breast carcinomas and melanomas. We and others have demonstrated that the KiSS1 peptide is a ligand for a specific G-protein coupled receptor. G-protein coupled receptors (GPCRs) represent important specific targets for a number of human diseases, ranging from the control of blood pressure, hormonal disorders, neurological diseases, to tumorigenesis and metastasis. Activation of KiSSl G-protein coupled receptor (KiSS1 GPCR) leads to the inhibition of cell proliferation, migration, and invasion in our preliminary in vitro experiments. However, the roles of KiSSl together with its cognate GPCR in tumor progression and metastasis are poorly understood and the signaling mechanisms of KiSSl and its GPCR have not been elucidated. Therefore, the long-term objectives of our research are to understand the functions of KiSSl, KiSSl GPCR, and its signaling pathways in tumor progression and metastasis; and to determine whether KiSSl and its GPCR can be used as a drug candidate and molecular target, respectively, in the treatment of human cancer metastasis. In this application, our 3 specific aims are: 1) Determine the roles of KiSSl and KiSSl GPCR in the complex processes of breast cancer metastasis including cell migration, invasion, tumorigenesis, and metastasis using in vitro cell culture systems, syngeneic and experimental breast tumor metastasis models; 2) Understand the in vivo function of KiSSl and its GPCR in breast cancer progression and metastasis using transgenic mouse models that overexpress KiSSl and its GPCR in mammary gland epithelial cells; 3) Determine the signaling mechanism of KiSSl and KiSSl GPCR mediated functions in tumor metastasis, especially the Rho family GTPases and their signaling pathways. Results obtained from these studies will not only elucidate the biological function of KiSSl and its GPCR in breast tumor progression and metastasis, but also reveal how KiSSl and its GPCR directly regulate signaling pathways in cell migration and invasion. The long-term benefit of this research is the great potential of KiSSl and its GPCR as an important drug candidate and molecular target for anti-cancer drug discovery and tumor metastasis treatment.

DESCRIPTION (provided by applicant): G-protein coupled receptors (GPCRs) and signal transduction pathways represent important specific targets for a variety of human diseases, ranging from the control of blood pressure, allergic response, hormonal disorders, to human cancer. We have recently identified a subfamily of prostate-specific G-protein coupled receptors (PSGRs). Although these specific receptors shares sequence homology with olfactory GPCRs in their seven putative transmembrane domains, the expression of PSGRs is highly restricted to the human prostate gland and is increased dramatically in human prostate cancers compared to matched normal tissues. Aberrant expression of PSGRs correlates well with prostate tumorigenesis, suggesting important roles of PSGRs in prostate cancer development and progression. However, how these prostate specific G-protein coupled receptors regulate prostate tumor progression is still not clear, and the signaling pathways and genes/proteins regulated by PSGRs have not been identified and characterized. Therefore, our long-term objectives are to understand the functions and signaling pathways of these PSGRs;and to determine whether PSGRs can be used as biomarkers in diagnosis and as molecular targets in the treatment of prostate cancers. We will test the hypothesis that overexpression of PSGRs will lead to the activation of specific growth signaling pathways to promote prostate tumor development and progression. Three specific aims are proposed in this grant application: 1) Determine the direct correlations of PSGR overexpression with prostate cancer development using orthotopic and transgenic mouse models;2) Delineate the mechanism of action for prostate-specific G-protein coupled receptors in prostate cancer development and progression;3) Determine the cooperative functions of PSGR overexpression and the loss of PTEN tumor suppressor gene in prostate tumor progression and metastasis. Results obtained from this study will provide information on the physiological functions of these prostate-specific G-protein coupled receptors in cancer development and progression and their potential uses as molecular targets in prostate cancer therapy. PUBLIC HEALTH RELEVANCE: The prostate-specific G-protein coupled receptors (PSGRs) are a novel subfamily of specific receptors identified in our lab and are dramatically increased in prostate cancer. Overexpression of PSGRs promotes cancer cell proliferation and tumorigenesis. Our long term goals are to understand how these prostate-specific receptors and their signaling pathways regulate prostate cancer development and progression;and to determine whether PSGRs can be used as biomarkers and molecular targets in the diagnosis and treatment of prostate cancers.