sphingosine-1-phosphate and Carcinoma

Hypoxia facilitates an aggressive phenotype and immune evasion in solid tumors including bladder cancer (BC). Sphingosine kinase 1 (SphK1) is aberrantly expressed and correlated with poor prognosis in BC patients. However, its roles in hypoxia-evoked malignancies and immune evasion in BC remain elusive.. The expression of SphK1 in BC tissues was analysed using a bioinformatics database. BC cells were transfected with si-SphK1 or recombinant HIF-1α plasmids under hypoxic conditions. The mRNA level, activity and protein expression of SphK1 were determined. Transwell assay was performed to evaluate cell invasion. After co-culture with natural killer (NK) cells, NK cell cytotoxicity to BC cells was assessed. The involvement of sphingosine-1-phosphate (S1P)/HIF-1α signaling was analysed by ELISA, qRT-PCR and western blot.. UALCAN and GEPIA database confirmed high expression of SphK1 in BC tissues. Moreover, hypoxia increased the expression and activity of SphK1. Loss of SphK1 inhibited hypoxia-induced cell invasion. IL-2 induced NK cell activation by secreting TNF-α and IFN-γ. Hypoxia antagonized NK cell activation-evoked cytotoxicity to BC cells. Intriguingly, SphK1 knockdown reversed hypoxia-induced cell resistance to NK cell killing. Mechanically, SphK1 loss inhibited hypoxia-activated the S1P/HIF-1α signaling. However, S1P addition reversed the inhibitory effects of SphK1 down-regulation on hypoxia-activated S1P/HIF-1α signaling. Notably, reactivating HIF-1α overturned the suppressive roles of SphK1 loss in decreasing hypoxia-induced cell invasion and resistance to NK cell cytotoxicity.. Targeting SphK1 may inhibit hypoxia-evoked invasion and immune evasion via the S1P/HIF-1α signaling, indicating a promising therapeutic target for BC.

Topics: Carcinoma; Cell Death; Humans; Hypoxia; Interleukin-2; Killer Cells, Natural; Oncogenes; Phosphotransferases (Alcohol Group Acceptor); RNA, Messenger; Tumor Necrosis Factor-alpha; Urinary Bladder; Urinary Bladder Neoplasms

Undifferentiated nasopharyngeal carcinoma (NPC) is a cancer with high metastatic potential that is consistently associated with Epstein-Barr virus (EBV) infection. In this study, we have investigated the functional contribution of sphingosine-1-phosphate (S1P) signalling to the pathogenesis of NPC. We show that EBV infection or ectopic expression of the EBV-encoded latent genes (EBNA1, LMP1, and LMP2A) can up-regulate sphingosine kinase 1 (SPHK1), the key enzyme that produces S1P, in NPC cell lines. Exogenous addition of S1P promotes the migration of NPC cells through the activation of AKT; shRNA knockdown of SPHK1 resulted in a reduction in the levels of activated AKT and inhibition of cell migration. We also show that S1P receptor 3 (S1PR3) mRNA is overexpressed in EBV-positive NPC patient-derived xenografts and a subset of primary NPC tissues, and that knockdown of S1PR3 suppressed the activation of AKT and the S1P-induced migration of NPC cells. Taken together, our data point to a central role for EBV in mediating the oncogenic effects of S1P in NPC and identify S1P signalling as a potential therapeutic target in this disease. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Topics: Adult; Aged; Animals; Carcinoma; Cell Line, Tumor; Cell Movement; Epstein-Barr Virus Infections; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Heterografts; Humans; Lysophospholipids; Male; Middle Aged; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Oncogene Protein v-akt; Receptors, Lysosphingolipid; RNA, Messenger; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Up-Regulation

We previously reported that sphingosine kinase 1 (SphK1), an enzyme that catalyzes the production of sphingosine-1-phosphate (SIP), is upregulated in human gastric cancer and predicts poor clinical outcome. In the present study, we used known differential effects of UV irradiation on human MGC-803 gastric cancer cells to determine their effect on SphK1 activity. Ectopic expression of SphK1 in MGC-803 gastric cancer cells markedly enhanced their resistance to UV irradiation, whereas silencing endogenous SphK1 with shRNAs weakened this ability. Furthermore, these anti-apoptotic effects were significantly associated with decrease of Bim, an apoptosis-related protein. We further demonstrated that SphK1 could downregulate the transcriptional activity of forkhead box O3a (FoxO3a) by inducing its phosphorylation, which was found to be associated with the PI3K/Akt signaling. Taken together, our study supports the theory that SphK1 confers resistance to apoptosis in gastric cancer cells via the Akt/FoxO3a/Bim pathway.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Carcinoma; Cell Line, Tumor; Down-Regulation; Forkhead Box Protein O3; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Lysophospholipids; Membrane Proteins; Phosphatidylinositol 3-Kinases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Signal Transduction; Sphingosine; Stomach Neoplasms; Ultraviolet Rays; Up-Regulation

Sphingosine 1-phosphate (S1P) induces migration of the human thyroid follicular carcinoma cell line ML-1 by activation of S1P(1) and S1P(3) receptors, G(i) proteins, and the phosphatidylinositol 3-kinase-Akt pathway. Because sphingosine kinase isoform 1 (SK) recently has been implicated as an oncogene in various cancer cell systems, we investigated the functions of SK in the migration, proliferation and adhesion of the ML-1 cell line. SK overexpressing ML-1 cells show an enhanced secretion of S1P, which can be attenuated, by inhibiting SK activity and a multidrug-resistant transport protein (ATP-binding cassette transporter). Furthermore, overexpression of SK enhances serum-induced migration of ML-1 cells, which can be attenuated by blocking ATP-binding cassette transporter and SK, suggesting that the migration is mediated by autocrine signaling through secretion of S1P. Inhibition of protein kinase C alpha, with both small interfering RNA (siRNA) and small molecular inhibitors attenuates migration in SK overexpressing cells. In addition, SK-overexpressing cells show an impaired adhesion, slower cell growth, and an up-regulation of ERK1/2 phosphorylation, as compared with cells expressing a dominant-negative SK. Taken together, we present evidence suggesting that SK enhances migration of ML-1 cells by an autocrine mechanism and that the S1P-evoked migration is dependent on protein kinase C alpha, ERK1/2, and SK.

Topics: Autocrine Communication; Carcinoma; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cells, Cultured; Humans; Lysophospholipids; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Invasiveness; Oncogenes; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C-alpha; RNA, Small Interfering; Sphingosine; Thyroid Neoplasms; Transfection

We previously demonstrated the regulation of epithelial ovarian cancer (EOC) cell invasiveness by the bioactive phospholipid sphingosine 1-phosphate (S1P). Low-dose S1P stimulated invasion like lysophosphatidic acid (LPA), while high-dose S1P inhibited invasion. Here we investigate how cell attachment status affects response to S1P and examine the effects of S1P and LPA on cell-cell and cell-extracellular matrix (ECM) adhesion.. EOC Dov13 cell invasion, ECM attachment and cell adhesion were tested through in vitro assays of Matrigel invasion and attachment to Matrigel, collagen or cell monolayer. Fractionated membrane and cytoplasmic proteins and biotin-labeled surface proteins were analyzed by western analysis. Actin cytoskeleton and FAK were visualized by immunofluorescence.. S1P (20 muM) inhibited invasion of sustained, attached cells but enhanced that of invading cells. Membrane N-cadherin was depleted upon reattachment to ECM. S1P pretreatment (20 muM) accelerated N-cadherin recovery, while 40 muM LPA or 0.5 muM S1P delayed recovery. Cell-cell adhesion and stress fibers were decreased by LPA and by 0.5 muM S1P but increased by 20 muM S1P. While S1P increased cellular attachment to Matrigel and collagen-I, LPA inhibited attachment to Matrigel. Surface N-cadherin, gamma- and beta-catenins, FAK and integrinbeta1 were altered by both reattachment and treatment with S1P or LPA.. S1P inversely affects invasion of attached and invading cells, switching from inhibition to stimulation. This switch is associated with depletion of N-cadherin and membrane FAK. The recovery of membrane N-cadherin, change in cell-cell adhesion and actin stress fibers intensity in response to LPA and S1P inversely correlate with their effects on cellular invasiveness.

Topics: Actins; Biocompatible Materials; Blotting, Western; Cadherins; Carcinoma; Cell Adhesion; Cell Line, Tumor; Collagen; Drug Combinations; Extracellular Matrix; Female; Fluorescent Antibody Technique; Focal Adhesion Kinase 1; Humans; Laminin; Lysophospholipids; Neoplasm Invasiveness; Ovarian Neoplasms; Proteoglycans; Sphingosine

Sphingosine-1-phosphate (SPP) induces a variety of cellular responses, including Ca2+ signaling, proliferation, and inhibition of motility, apparently by acting at specific G protein coupled receptors. Here, the expression, signaling, and motile responses of sphingolipid receptors were examined in human bladder carcinoma (J82) cells, for which lysophosphatidic acid (LPA) and thrombin act as potent agonists. SPP potently and rapidly mobilized Ca2+, stimulated phospholipases C and D, and inhibited cAMP accumulation, without affecting growth of J82 cells, which express the recently identified SPP receptors, Edg-1 and Edg-3. The effects of SPP were mimicked by sphingosylphosphorylcholine (SPPC) and strongly attenuated by pertussis toxin (PTX). SPP and SPPC by themselves induced a small, PTX-sensitive motile response. However, stimulation of cell motility by LPA, which by itself was also PTX-sensitive, was blocked by SPP and SPPC. In contrast, motility stimulation by thrombin, which by itself was PTX-insensitive, was strongly augmented by the sphingolipids in a PTX-sensitive manner. The bidirectional regulation of LPA- and thrombin-stimulated motility was not due to selective alterations in the activation of Rho GTPases which control cell motility. In fact, RhoA activation and Rho-dependent actin stress fiber formation induced by LPA and thrombin were mimicked, but not altered by SPP and SPPC. We conclude that J82 cells express sphingolipid receptors, coupled via G proteins to several signaling pathways. Most importantly, these sphingolipid receptors potently regulate thrombin- and LPA-stimulated motility, but in opposite directions, suggesting that migration of these human bladder carcinoma cells is controlled by a complex network of interacting extracellular ligands.

Topics: Actins; Calcium; Carcinoma; Cell Division; Cell Movement; Cyclic AMP; GTP-Binding Proteins; Humans; Lysophospholipids; Phospholipase D; Phosphorylcholine; Receptors, Cell Surface; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Sphingolipids; Sphingosine; Thrombin; Tumor Cells, Cultured; Type C Phospholipases; Urinary Bladder Neoplasms

Our recent studies have suggested that sphingosine, an endogenous protein kinase C (PKC) inhibitor, may mediate apoptosis induced by a phorbol ester (PMA) in human promyelocytic leukemia HL-60 cells [Ohta et al. Cancer Res. 1995;55:691-697], and that the apoptotic induction by both PMA and sphingosine is accompanied by down-regulation of bcl-2, a gene which acts to prevent apoptotic cell death [Sakakura et al. FEBS Lett. 1996;397:177-180]. In this study, we examined the sphingosine-induced apoptosis of the androgen-independent human prostatic carcinoma cell line DU-145, which expresses bcl-X(L) and Bax but not bcl-2, and found that treatment of DU-145 cells with sphingosine suppressed bcl-X(L) in both mRNA and protein levels but did not change bax expression at all. In contrast, in apoptotic cells treated with a PKC inhibitor, staurosporine, no effect on bcl-X(L) or bax expression was observed. The initial metabolites of sphingosine in the cells, ceramide and sphingosine 1-phosphate, failed to induce apoptosis. These results indicate that, in DU-145 cells, sphingosine, but not its metabolites, induces apoptosis through down-regulation of bcl-X(L), independently of PKC inhibition. Our present results, together with previous observations, strongly suggest that apoptosis regulatory genes differ according to cell type and apoptosis induction through sphingosine is accompanied by inhibition of either bcl-2 or bcl-X(L) activity in these cells.

Topics: Androgens; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Carcinoma; Down-Regulation; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Lysophospholipids; Male; Prostatic Neoplasms; Protein Kinase C; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Sphingosine; Staurosporine