James R. Van Brocklyn

MicroRNA-451 Regulates LKB1/AMPK Signaling and Allows Adaptation to Metabolic Stress in Glioma Cells

To sustain tumor growth, cancer cells must be able to adapt to fluctuations in energy availability. We have identified a single microRNA that controls glioma cell proliferation, migration, and responsiveness to glucose deprivation. Abundant glucose allows relatively high miR-451 expression, promoting cell growth. In low glucose, miR-451 levels decrease, slowing proliferation but enhancing migration and survival. This allows cells to survive metabolic stress and seek out favorable growth conditions. In glioblastoma patients, elevated miR-451 is associated with shorter survival. The effects of miR-451 are mediated by LKB1, which it represses through targeting its binding partner, CAB39 (MO25 alpha). Overexpression of miR-451 sensitized cells to glucose deprivation, suggesting that its downregulation is necessary for robust activation of LKB1 in response to metabolic stress. Thus, miR-451 is a regulator of the LKB1/AMPK pathway, and this may represent a fundamental mechanism that contributes to cellular adaptation in response to altered energy availability.

Sphingosine kinase-1 expression correlates with poor survival of patients with glioblastoma multiforme: roles of sphingosine kinase isoforms in growth of glioblastoma cell lines.

Sphingosine-1-phosphate is a bioactive lipid that is mitogenic for human glioma cell lines by signaling through its G protein-coupled receptors. We investigated the role of sphingosine-1-phosphate receptors and the enzymes that form sphingosine-1-phosphate, sphingosine kinase (SphK)-1, and -2 in human astrocytomas. Astrocytomas of various histologic grades expressed three types of sphingosine-1-phosphate receptors, S1P1, S1P2, and S1P3; however, no significant correlation with histologic grade or patient survival was detected. Expression of SphK1, but not SphK2, in human astrocytoma grade 4 (glioblastoma multiforme) tissue correlated with short patient survival. Patients whose tumors had low SphK1 expression survived a median 357 days, whereas those with high levels of SphK1 survived a median 102 days. Decreasing SphK1 expression using RNA interference or pharmacologic inhibition of SphK significantly decreased the rate of proliferation of U-1242 MG and U-87 MG glioblastoma cell lines. Surprisingly, RNA interference to knockdown SphK2 expression inhibited glioblastoma cell proliferation more potently than did SphK1 knockdown. SphK knockdown also prevented cells from exiting G1 phase of the cell cycle and marginally increased apoptosis. Thus, SphK isoforms may be major contributors to growth of glioblastoma cells in vitro and to aggressive behavior of glioblastoma multiforme.

Identification of Edg1 receptor residues that recognize sphingosine 1-Phosphate

Originating from its DNA sequence, a computational model of the Edg1 receptor has been developed that predicts critical interactions with its ligand, sphingosine 1-phosphate. The basic amino acids Arg120 and Arg292 ion pair with the phosphate, whereas the acidic Glu121 residue ion pairs with the ammonium moiety of sphingosine 1-phosphate. The requirement of these interactions for specific ligand recognition has been confirmed through examination of site-directed mutants by radioligand binding, ligand-induced [35S]GTPγS binding, and receptor internalization assays. These ion-pairing interactions explain the ligand specificity of the Edg1 receptor and provide insight into ligand specificity differences within the Edg receptor family. This computational map of the ligand binding pocket provides information necessary for understanding the molecular pharmacology of this receptor, thus underlining the potential of the computational method in predicting ligand-receptor interactions.

The control of the balance between ceramide and sphingosine-1-phosphate by sphingosine kinase: oxidative stress and the seesaw of cell survival and death.

Sphingolipids are components of all eukaryotic cells that play important roles in a wide variety of biological processes. Ceramides and sphingosine-1-phosphate (S1P) are signaling molecules that regulate cell fate decisions in a wide array of species including yeast, plants, vertebrates, and invertebrates. Ceramides favor anti-proliferative and cell death pathways such as senescence and apoptosis, whereas S1P stimulates cell proliferation and survival pathways. The control of cell fate by these two interconvertible lipids has been called the sphingolipid rheostat or sphingolipid biostat. Sphingosine kinase, the enzyme that synthesizes S1P, is a crucial enzyme in regulation of the balance of these sphingolipids. Sphingosine kinase has been shown to play dynamic roles in the responses of cells to stress, leading to modulation of cell fate through a variety of signaling pathways impinging on the processes of cell proliferation, apoptosis, autophagy and senescence. This review summarizes the roles of sphingosine kinase signaling in these processes and the mechanisms mediating these responses. In addition, we discuss the evidence tying sphingosine kinase-mediated stress responses to the process of aging.

Sphingosine-1-phosphate stimulates motility and invasiveness of human glioblastoma multiforme cells

Sphingosine-1-phosphate (S1P) is a bioactive lipid which is a potent mitogen for glioblastoma multiforme cells. Here we show that S1P also potently enhances the in vitro motility of glioblastoma cells by signaling through receptors coupled to G(i/o) proteins. Moreover, S1P also enhanced in vitro invasion of glioblastoma cells through Matrigel. S1P had no effect on matrix metalloproteinase secretion but did enhance glioblastoma cell adhesion. S1P is present at high levels in brain tissue. Thus it is possible that autocrine or paracrine signaling by S1P through its G protein-coupled receptors enhances both glioma cell proliferation and invasiveness.

Sphingosine-1-phosphate stimulates human glioma cell proliferation through Gi-coupled receptors: role of ERK MAP kinase and phosphatidylinositol 3-kinase β

The regulation of glioma cell proliferation by sphingosine-1-phosphate (S1P) was studied using the human glioblastoma cell line U-373 MG. U-373 MG cells responded mitogenically to nanomolar concentrations of S1P, and express mRNA encoding the S1P receptors S1P1/endothelial differentiation gene (EDG)-1, S1P3/EDG-3 and S1P2/EDG-5. S1P-induced proliferation required extracellular signal-regulated kinase activation and was partially sensitive to pertussis toxin and wortmannin, indicating involvement of a Gi-coupled receptor and phosphatidylinositol 3-kinase. Moreover, S1P1, S1P3 and S1P2 receptors are expressed in the majority of human glioblastomas as determined by reverse transcriptase-polymerase chain reaction analysis. Thus, S1P signaling through EDG receptors may contribute to glioblastoma growth in vivo.

Sphingosine-1-Phosphate Regulates Glioblastoma Cell Invasiveness Through The Urokinase Plasminogen Activator System and CCN1/Cyr61

Glioblastoma multiforme (GBM) is an aggressively invasive brain neoplasm with poor patient prognosis. We have previously shown that the bioactive lipid sphingosine-1-phosphate (S1P) stimulates in vitro invasiveness of GBM cells and that high expression levels of the enzyme that forms S1P, sphingosine kinase-1 (SphK1), correlate with shorter survival time of GBM patients. We also recently showed that S1P induces expression of CCN1 (also known as Cyr61), a matricellular protein known to correlate with poor patient prognosis, in GBM cells. In this study, we further explored the role of CCN1 as well as the urokinase plasminogen activator (uPA), a protein known to stimulate GBM cell invasiveness, in S1P-induced invasion using a spheroid invasion assay. We also investigated the roles of various S1P receptors in stimulating invasiveness through these pathways. S1P induced expression of uPA and its receptor, uPAR, in GBM cells. Whereas S1P1, S1P2, and S1P3 receptors all contribute, at least partially, S1P1 overexpression led to the most dramatic induction of the uPA system and of spheroid invasion, even in the absence of added S1P. Furthermore, neutralizing antibodies directed against uPA or CCN1 significantly decreased both basal and S1P-stimulated GBM cell invasiveness. Inhibition of SphK blocked basal expression of uPA and uPAR, as well as glioma cell invasion; however, overexpression of SphK did not augment S1P receptor–mediated enhancement of uPA activity or invasion. Thus, SphK is necessary for basal activity of the uPA system and glioma cell invasion, whereas S1P receptor signaling enhances invasion, partially through uPA and CCN1. (Mol Cancer Res 2009;7(1):23–32)

Text mining of full-text journal articles combined with gene expression analysis reveals a relationship between sphingosine-1-phosphate and invasiveness of a glioblastoma cell line

BackgroundSphingosine 1-phosphate (S1P), a lysophospholipid, is involved in various cellular processes such as migration, proliferation, and survival. To date, the impact of S1P on human glioblastoma is not fully understood. Particularly, the concerted role played by matrix metalloproteinases (MMP) and S1P in aggressive tumor behavior and angiogenesis remains to be elucidated.ResultsTo gain new insights in the effect of S1P on angiogenesis and invasion of this type of malignant tumor, we used microarrays to investigate the gene expression in glioblastoma as a response to S1P administration in vitro. We compared the expression profiles for the same cell lines under the influence of epidermal growth factor (EGF), an important growth factor. We found a set of 72 genes that are significantly differentially expressed as a unique response to S1P. Based on the result of mining full-text articles from 20 scientific journals in the field of cancer research published over a period of five years, we inferred gene-gene interaction networks for these 72 differentially expressed genes. Among the generated networks, we identified a particularly interesting one. It describes a cascading event, triggered by S1P, leading to the transactivation of MMP-9 via neuregulin-1 (NRG-1), vascular endothelial growth factor (VEGF), and the urokinase-type plasminogen activator (uPA). This interaction network has the potential to shed new light on our understanding of the role played by MMP-9 in invasive glioblastomas.ConclusionAutomated extraction of information from biological literature promises to play an increasingly important role in biological knowledge discovery. This is particularly true for high-throughput approaches, such as microarrays, and for combining and integrating data from different sources. Text mining may hold the key to unraveling previously unknown relationships between biological entities and could develop into an indispensable instrument in the process of formulating novel and potentially promising hypotheses.

Signal transduction of sphingosine-1-phosphate G protein-coupled receptors.

Sphingosine-1-phosphate (S1P) is a bioactive lipid capable of eliciting dramatic effects in a variety of cell types. Signaling by this molecule is by a family of five G protein—coupled receptors named S1P1–5 that signal through a variety of pathways to regulate cell proliferation, migration, cytoskeletal organization, and differentiation. These receptors are expressed in a wide variety of tissues and cell types, and their cellular effects contribute to important biological and pathological functions of S1P in many processes, including angiogenesis, vascular development, lymphocyte trafficking, and cancer. This review will focus on the current progress in the field of S1P receptor signaling and biology.

Ganglioside modulation of the PDGF receptor. A model for ganglioside functions.

SummaryGangliosides are a family of glycolipids that are present at the cell surface of all mammalian cells. Patterns of gangliosides are different in gliomas than normal brain, and exogenously added gangliosides affect the growth of cultured glioma cells. Gangliosides inhibit the activities of several kinases, including protein kinase C (PKC) and cAMP-kinase. U-1242 MG cells (derived from a human malignant glioma) have receptors for platelet-derived growth factor (PDGF) that become phosphorylated on tyrosine when exposed to PDGF. Exposure of these cells to PDGF also causes an increase in intracellular calcium concentration ([Ca2+]i) and induces a translocation of PKC to the membrane. Preincubation of U-1242 MG cells with several species of gangliosides inhibits the increase in ([Ca2+]i) and PKC translocation in response to PDGF, but GM3 is much less effective than other species tested. This is due to a lack of activation of the receptor tyrosine kinase as monitored by phosphorylation of the receptor on tyrosine residues, but is not due to an inhibition of binding of PDGF to its receptors. The lack of activation of the PDGF receptor tyrosine kinase is due to an inhibition of dimerization of the receptor monomers by gangliosides GM1, GM2, GD1a, GT1b, but not GM3. Therefore, gangliosides may be involved in coordinating the activities of multiple trophic factors simultaneously acting on a cell by regulating the dimerization of their respective receptor monomers.