Phosphorylation-dependent translocation of sphingosine kinase to the plasma membrane drives its oncogenic signalling

Sphingosine kinase (SK) 1 catalyzes the formation of the bioactive lipid sphingosine 1-phosphate, and has been implicated in several biological processes in mammalian cells, including enhanced proliferation, inhibition of apoptosis, and oncogenesis. Human SK (hSK) 1 possesses high instrinsic catalytic activity which can be further increased by a diverse array of cellular agonists. We have shown previously that this activation occurs as a direct consequence of extracellular signal–regulated kinase 1/2–mediated phosphorylation at Ser225, which not only increases catalytic activity, but is also necessary for agonist-induced translocation of hSK1 to the plasma membrane. In this study, we report that the oncogenic effects of overexpressed hSK1 are blocked by mutation of the phosphorylation site despite the phosphorylation-deficient form of the enzyme retaining full instrinsic catalytic activity. This indicates that oncogenic signaling by hSK1 relies on a phosphorylation-dependent function beyond increasing enzyme activity. We demonstrate, through constitutive localization of the phosphorylation-deficient form of hSK1 to the plasma membrane, that hSK1 translocation is the key effect of phosphorylation in oncogenic signaling by this enzyme. Thus, phosphorylation of hSK1 is essential for oncogenic signaling, and is brought about through phosphorylation-induced translocation of hSK1 to the plasma membrane, rather than from enhanced catalytic activity of this enzyme

Estrogen transactivates EGFR via the sphingosine 1-phosphate receptor Edg-3: the role of sphingosine kinase-1

The transactivation of enhanced growth factor receptor (EGFR) by G protein–coupled receptor (GPCR) ligands is recognized as an important signaling mechanism in the regulation of complex biological processes, such as cancer development. Estrogen (E2), which is a steroid hormone that is intimately implicated in breast cancer, has also been suggested to function via EGFR transactivation. In this study, we demonstrate that E2-induced EGFR transactivation in human breast cancer cells is driven via a novel signaling system controlled by the lipid kinase sphingosine kinase-1 (SphK1). We show that E2 stimulates SphK1 activation and the release of sphingosine 1-phosphate (S1P), by which E2 is capable of activating the S1P receptor Edg-3, resulting in the EGFR transactivation in a matrix metalloprotease–dependent manner. Thus, these findings reveal a key role for SphK1 in the coupling of the signals between three membrane-spanning events induced by E2, S1P, and EGF. They also suggest a new signal transduction model across three individual ligand-receptor systems, i.e., “criss-cross” transactivation

Novel miRNA-based drug CD5-2 reduces liver tumor growth in diethylnitrosamine-treated mice by normalizing tumor vasculature and altering immune infiltrate

IntroductionLiver cancers exhibit abnormal (leaky) vasculature, hypoxia and an immunosuppressive microenvironment. Normalization of tumor vasculature is an emerging approach to treat many cancers. Blockmir CD5-2 is a novel oligonucleotide-based inhibitor of the miR-27a interaction with VE-Cadherin, the endothelial-specific cadherin. The combination of a vasoactive medication with inhibition of immune checkpoints such as programmed cell death protein 1 (PD1) has been shown to be effective in treating liver cancer in humans. We aimed to study the effect of CD5-2 combined with checkpoint inhibition (using an antibody against PD1) on liver tumor growth, vasculature and immune infiltrate in the diethylnitrosamine (DEN)-induced liver tumor mouse model.MethodsWe first analyzed human miR-27a and VE-Cadherin expression data from The Cancer Genome Atlas for hepatocellular carcinoma. CD5-2 and/or anti-PD1 antibody were given to the DEN-treated mice from age 7-months until harvest at age 9-months. Tumor and non-tumor liver tissues were analyzed using histology, immunohistochemistry, immunofluorescence and scanning electron microscopy.ResultsHuman data showed high miR-27a and low VE-Cadherin were both significantly associated with poorer prognosis. Mice treated with CD5-2 plus anti-PD1 antibody had significantly smaller liver tumors (50% reduction) compared to mice treated with either agent alone, controls, or untreated mice. There was no difference in tumor number. Histologically, tumors in CD5-2-treated mice had less leaky vessels with higher VE-Cadherin expression and less tumor hypoxia compared to non-CD5-2-treated mice. Only tumors in the combination CD5-2 plus anti-PD1 antibody group exhibited a more favorable immune infiltrate (significantly higher CD3+ and CD8+ T cells and lower Ly6G+ neutrophils) compared to tumors from other groups.DiscussionCD5-2 normalized tumor vasculature and reduced hypoxia in DEN-induced liver tumors. CD5-2 plus anti-PD1 antibody reduced liver tumor size possibly by altering the immune infiltrate to a more immunosupportive one

The Protein Tyrosine Phosphatase Pez Is a Major Phosphatase of Adherens Junctions and Dephosphorylates β-Catenin

Cell-cell adhesion regulates processes important in embryonal development, normal physiology, and cancer progression. It is regulated by various mechanisms including tyrosine phosphorylation. We have previously shown that the protein tyrosine phosphatase Pez is concentrated at intercellular junctions in confluent, quiescent monolayers but is nuclear in cells lacking cell-cell contacts. We show here with an epithelial cell model that Pez localizes to the adherens junctions in confluent monolayers. A truncation mutant lacking the catalytic domain acts as a dominant negative mutant to upregulate tyrosine phosphorylation at adherens junctions. We identified β-catenin, a component of adherens junctions, as a substrate of Pez by a “substrate trapping” approach and by in vitro dephosphorylation with recombinant Pez. Consistent with this, ectopic expression of the dominant negative mutant caused an increase in tyrosine phosphorylation of β-catenin, demonstrating that Pez regulates the level of tyrosine phosphorylation of adherens junction proteins, including β-catenin. Increased tyrosine phosphorylation of adherens junction proteins has been shown to decrease cell-cell adhesion, promoting cell migration as a result. Accordingly, the dominant negative Pez mutant enhanced cell motility in an in vitro “wound” assay. This suggests that Pez is also a regulator of cell motility, most likely through its action on cell-cell adhesion

Assessing IRES activity in the HIF-1α and other cellular 5′ UTRs

Dicistronic reporter plasmids, such as the dual luciferase-containing pR-F plasmid, have been widely used to assay cellular and viral 5′ untranslated regions (UTRs) for IRES activity. We found that the pR-F dicistronic reporter containing the 5′ UTRs from HIF-1α, VEGF, c-myc, XIAP, VEGFR-1, or Egr-1 UTRs all produce the downstream luciferase predominantly as a result of cryptic promoter activity that is activated by the SV40 enhancer elements in the plasmid. RNA transfection experiments using dicistronic or uncapped RNAs, which avoid the complication of cryptic promoter activity, indicate that the HIF-1α, VEGF, c-myc, and XIAP UTRs do have some IRES activity, although the activity was much less than that of the viral EMCV IRES. The translation of transfected monocistronic RNAs containing these cellular UTRs was greatly enhanced by the presence of a 5′ cap, raising questions as to the strength or mechanism of IRES-mediated translation in these assays

Maturation In Vivo of Schistosoma mansoni Schistosomula After Culture In Vitro with Granulocytes and Antibody

Seven experiments were carried out to test the relationship between the morphological assay for damage to schistosomula in vitro with toluidine blue and the loss of the ability of damaged organisms to mature in vivo. Schistosomula were prepared by penetration of rat skin and cultured for 12 to 38 h in the presence of various combinations of purified human eosinophils or neutrophils and heat-inactivated human antischistosomular serum. Samples were scored for microscopically detectable damage, and the remaining organisms were injected intravenously into normal mice. These mice were perfused after 5.5 to 7 weeks, and the recovery of adult worms was determined. After culture of schistosomula in medium alone, between 8.4 and 32.7% of injected organisms matured into adult worms. There was no significant difference in the capacity of freshly prepared and cultured schistosomula to mature in vivo. Schistosomula cultured with antibody alone showed no significant damage in vitro, and in only one of seven experiments was there a significant (35%) reduction compared with the medium controls in their capacity to mature in vivo. Schistosomula cultured with neutrophils alone or eosinophils alone showed no significant damage in vitro and no loss of viability in vivo. Schistosomula cultured with neutrophils and antibody showed a 28% reduction in recovery in one experiment but an increase in recovery (12 and 46%) in two other experiments. In contrast, schistosomula cultured with eosinophils and antibody showed evidence of both marked damage in vitro (22 to 93% dead organisms) and loss of viability in vivo (26 to 98% reduction in recovery) in all seven experiments. These findings justify the use of the toluidine blue morphological assay as an estimate of irreversible damage to schistosomula and confirm that human eosinophils and neutrophils differ markedly in their capacity to mediate antibody-dependent damage in vitro