A selective ATP-competitive sphingosine kinase inhibitor demonstrates anti-cancer properties

The dynamic balance of cellular sphingolipids, the sphingolipid rheostat, is an important determinant of cell fate, and is commonly deregulated in cancer. Sphingosine 1-phosphate is a signaling molecule with anti-apoptotic, pro-proliferative and pro-angiogenic effects, while conversely, ceramide and sphingosine are pro-apoptotic. The sphingosine kinases (SKs) are key regulators of this sphingolipid rheostat, and are attractive targets for anti-cancer therapy. Here we report a first-in-class ATP-binding site-directed small molecule SK inhibitor, MP-A08, discovered using an approach of structural homology modelling of the ATP-binding site of SK1 and in silico docking with small molecule libraries. MP-A08 is a highly selective ATP competitive SK inhibitor that targets both SK1 and SK2. MP-A08 blocks pro-proliferative signalling pathways, induces mitochondrial-associated apoptosis in a SK-dependent manner, and reduces the growth of human lung adenocarcinoma tumours in a mouse xenograft model by both inducing tumour cell apoptosis and inhibiting tumour angiogenesis. Thus, this selective ATP competitive SK inhibitor provides a promising candidate for potential development as an anti-cancer therapy, and also, due to its different mode of inhibition to other known SK inhibitors, both validates the SKs as targets for anti-cancer therapy, and represents an important experimental tool to study these enzymes.Melissa R. Pitman, Jason A. Powell, Carl Coolen, Paul A.B. Moretti, Julia R. Zebol, Duyen H. Pham, John W. Finnie, Anthony S. Don, Lisa M. Ebert, Claudine S. Bonder, Briony L. Gliddon, Stuart M. Pitso

Reduction in sphingosine kinase 1 influences the susceptibility to dengue virus infection by altering antiviral responses

Published Online: 01/01/2016Sphingosine kinase (SK) 1 is a host kinase that enhances some viral infections. Here we investigated the ability of SK1 to modulate dengue virus (DENV) infection in vitro. Overexpression of SK1 did not alter DENV infection; however, targeting SK1 through chemical inhibition resulted in reduced DENV RNA and infectious virus release. DENV infection of SK1⁻/ ⁻ murine embryonic fibroblasts (MEFs) resulted in inhibition of infection in an immortalized line (iMEF) but enhanced infection in primary MEFs (1°MEFs). Global cellular gene expression profiles showed expected innate immune mRNA changes in DENV-infected WT but no induction of these responses in SK1⁻/⁻  iMEFs. Reverse transciption PCR demonstrated a low-level induction of IFN-β and poor induction of mRNA for the interferon-stimulated genes (ISGs) viperin, IFIT1 and CXCL10 in DENV-infected SK1⁻/⁻  compared with WT iMEFs. Similarly, reduced induction of ISGs was observed in SK1⁻/⁻  1°MEFs, even in the face of high-level DENV replication. In both iMEFs and 1°MEFs, DENV infection induced production of IFN-β protein. Additionally, higher basal levels of antiviral factors (IRF7, CXCL10 and OAS1) were observed in uninfected SK1⁻/⁻  iMEFs but not 1°MEFs. This suggests that, in this single iMEF line, lack of SK1 upregulates the basal levels of factors that may protect cells against DENV infection. More importantly, regardless of the levels of DENV replication, all cells that lacked SK1 produced IFN-β but were refractory to induction of ISGs such as viperin, IFIT1 and CXCL10. Based on these findings, we propose new roles for SK1 in affecting innate responses that regulate susceptibility to DENV infection.Jennifer N. Clarke, Lorena K. Davies, Julie K. Calvert, Briony L. Gliddon, Wisam H. Al Shujari, Amanda L. Aloia, Karla J. Helbig, Michael R. Beard, Stuart M. Pitson and Jillian M. Car

Sphingosine 1-phosphate is a ligand for peroxisome proliferator-activated receptor-gamma that regulates neoangiogenesis

Sphingosine 1-phosphate (S1P) is a bioactive lipid that can function both extracellularly and intracellularly to mediate a variety of cellular processes. Using lipid affinity matrices and a radiolabeled lipid binding assay, we reveal that S1P directly interacts with the transcription factor peroxisome proliferator-activated receptor (PPAR)γ. Herein, we show that S1P treatment of human endothelial cells (ECs) activated a luciferase-tagged PPARγ-specific gene reporter by ∼12-fold, independent of the S1P receptors. More specifically, in silico docking, gene reporter, and binding assays revealed that His323 of the PPARγ ligand binding domain is important for binding to S1P. PPARγ functions when associated with coregulatory proteins, and herein we identify that peroxisome proliferator-activated receptor-γ coactivator 1 (PGC1)β binds to PPARγ in ECs and their progenitors (nonadherent endothelial forming cells) and that the formation of this PPARγ:PGC1β complex is increased in response to S1P. ECs treated with S1P selectively regulated known PPARγ target genes with PGC1β and plasminogen-activated inhibitor-1 being increased, no change to adipocyte fatty acid binding protein 2 and suppression of CD36. S1P-induced in vitro tube formation was significantly attenuated in the presence of the PPARγ antagonist GW9662, and in vivo application of GW9662 also reduced vascular development in Matrigel plugs. Interestingly, activation of PPARγ by the synthetic ligand troglitazone also reduced tube formation in vitro and in vivo. To support this, Sphk1(-/-)Sphk2(+/-) mice, with low circulating S1P levels, demonstrated a similar reduction in vascular development. Taken together, our data reveal that the transcription factor, PPARγ, is a bona fide intracellular target for S1P and thus suggest that the S1P:PPARγ:PGC1β complex may be a useful target to manipulate neovascularization.Kate A. Parham, Julia R. Zebol, Katie L. Tooley, Wai Y. Sun, Lachlan M. Moldenhauer, Michaelia P. Cockshell, Briony L. Gliddon, Paul A. Moretti, Gabor Tigyi, Stuart M. Pitson, and Claudine S. Bonde

CIB2 negatively regulates oncogenic signaling in ovarian cancer via sphingosine kinase 1

Abstract not availableWenying Zhu, Kate E. Jarman, Noor A. Lokman, Heidi A. Neubauer, Lorena T. Davies, Briony L. Gliddon, Houng Taing, Paul A.B. Moretti, Martin K. Oehler, Melissa R. Pitman, and Stuart M. Pitso

CIB1 contributes to oncogenic signalling by Ras via modulating the subcellular localisation of sphingosine kinase 1

CIB1 (calcium and integrin binding protein 1) is a small intracellular protein with numerous interacting partners, and hence has been implicated in various cellular functions. Recent studies have revealed emerging roles of CIB1 in regulating cancer cell survival and angiogenesis, although the mechanisms involved have remained largely undefined. In investigating the oncogenic function of CIB1, we initially found that CIB1 is widely up-regulated across a diverse range of cancers, with this upregulation frequently correlating with oncogenic mutations of KRas. Consistent with this, we found that ectopic expression of oncogenic KRas and HRas in cells resulted in elevated CIB1 expression. We previously described the Ca²⁺-myristoyl switch function of CIB1, and its ability to facilitate agonist-induced plasma membrane localisation of sphingosine kinase 1 (SK1), a location where SK1 is known to elicit oncogenic signalling. Thus, we examined the role this may play in oncogenesis. Consistent with these findings, we demonstrated here that over-expression of CIB1 by itself is sufficient to drive localisation of SK1 to the plasma membrane and enhance the membrane-associated enzymatic activity of SK1, as well as its oncogenic signalling. We subsequently demonstrated that elevated levels of CIB1 resulted in full neoplastic transformation, in a manner dependent on SK1. In agreement with our previous findings that SK1 is a downstream mediator of oncogenic signalling by Ras, we found that targeting CIB1 also inhibited neoplastic growth of cells induced by oncogenic Ras, suggesting an important pro-tumorigenic role for CIB1. Thus, we have demonstrated for the first time a role for CIB1 in neoplastic transformation, and revealed a novel mechanism facilitating oncogenic signalling by Ras and SK1.W Zhu, BL Gliddon, KE Jarman, PAB Moretti, T Tin, LV Parise, JM Woodcock, JA Powell, A Ruszkiewicz, MR Pitman, and SM Pitso

A randomized head to head trial of MoodSwings.net.au: an internet based self-help program for bipolar disorder.

Adjunctive psychosocial interventions are efficacious in bipolar disorder, but their incorporation into routine management plans are often confounded by cost and access constraints. We report here a comparative evaluation of two online programs hosted on a single website (www.moodswings.net.au). A basic version, called MoodSwings (MS), contains psychoeducation material and asynchronous discussion boards; and a more interactive program, MoodSwings Plus (MS-Plus), combined the basic psychoeducation material and discussion boards with elements of Cognitive Behavioral Therapy. These programs were evaluated in a head-to-head study design

Rapid disruption of axon-glial integrity in response to mild cerebral hypoperfusion

Myelinated axons have a distinct protein architecture essential for action potential propagation, neuronal communication, and maintaining cognitive function. Damage to myelinated axons, associated with cerebral hypoperfusion, contributes to age-related cognitive decline. We sought to determine early alterations in the protein architecture of myelinated axons and potential mechanisms after hypoperfusion. Using a mouse model of hypoperfusion, we assessed changes in proteins critical to the maintenance of paranodes, nodes of Ranvier, axon–glial integrity, axons, and myelin by confocal laser scanning microscopy. As early as 3 d after hypoperfusion, the paranodal septate-like junctions were damaged. This was marked by a progressive reduction of paranodal Neurofascin signal and a loss of septate-like junctions. Concurrent with paranodal disruption, there was a significant increase in nodal length, identified by Nav1.6 staining, with hypoperfusion. Disruption of axon–glial integrity was also determined after hypoperfusion by changes in the spatial distribution of myelin-associated glycoprotein staining. These nodal/paranodal changes were more pronounced after 1 month of hypoperfusion. In contrast, the nodal anchoring proteins AnkyrinG and Neurofascin 186 were unchanged and there were no overt changes in axonal and myelin integrity with hypoperfusion. A microarray analysis of white matter samples indicated that there were significant alterations in 129 genes. Subsequent analysis indicated alterations in biological pathways, including inflammatory responses, cytokine-cytokine receptor interactions, blood vessel development, and cell proliferation processes. Our results demonstrate that hypoperfusion leads to a rapid disruption of key proteins critical to the stability of the axon–glial connection that is mediated by a diversity of molecular events