sphingosine-kinase and Cell-Transformation--Neoplastic

The various sphingosine kinase (SphK) isoenzymes (isozymes) and isoforms, key players in normal cellular physiology, are strongly implicated in cancer and other diseases. Mutations in SphKs, that may justify abnormal physiological function, have not been recorded. Nonetheless, there is a large and growing body of evidence demonstrating the contribution of gain or loss of function and the imbalance in the SphK/S1P rheostat to a plethora of pathological conditions including cancer, diabetes and inflammatory diseases. SphK is expressed as two isozymes SphK1 and SphK2, transcribed from genes located on different chromosomes and both isozymes catalyze the phosphorylation of sphingosine to S1P. Expression of each SphK isozyme produces alternately spliced isoforms. In recent years the importance of the contribution of SpK1 expression to treatment resistance in cancer has been highlighted and, additionally, differences in treatment outcome appear to also be dependent upon SphK isoform expression. This review focuses on an exciting emerging area of research involving SphKs functions, expression and subcellular localization, highlighting the complexity of targeting SphK in cancer and also comorbid diseases. This review also covers the SphK isoenzymes and isoforms from a historical perspective, from their first discovery in murine species and then in humans, their role(s) in normal cellular function and in disease processes, to advancement of SphK as an oncotarget.

Topics: Animals; Biomarkers, Tumor; Cell Transformation, Neoplastic; Disease Susceptibility; Drug Discovery; Drug Resistance, Neoplasm; Gene Expression Regulation; Humans; Isoenzymes; Mice; Molecular Targeted Therapy; Multigene Family; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Proprotein Convertases; Protein Binding; Receptors, Lysosphingolipid; Serine Endopeptidases

Topics: Animals; Antineoplastic Agents; Cell Transformation, Neoplastic; Ceramides; Humans; Lysophospholipids; Molecular Targeted Therapy; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysophospholipid; Signal Transduction; Sphingosine

Sphingosine-1-phosphate (S1P) signaling has been widely explored as a therapeutic target in cancer. Sphingosine kinase 2 (SK2), one of the kinases that phosphorylate sphingosine, has a cell type and cell location-dependent mechanism of action, so the ability of SK2 to induce cell cycle arrest, apoptosis, proliferation, and survival is strongly influenced by the cell-context. In contrast to SK1, which is widely studied in different types of cancer, including head and neck cancer, the role of SK2 in the development and progression of oral cancer is still poorly understood. In order to elucidate SK2 role in oral cancer, we performed the overexpression of SK2 in non-tumor oral keratinocyte cell (NOK SK2) and in oral squamous cell carcinoma (HN12 SK2), and RNA interference for SK2 in another oral squamous cell carcinoma (HN13 shSK2). In our study we demonstrate for the first time that accumulation of SK2 can be a starting point for oncogenesis and transforms a non-tumor oral keratinocyte (NOK-SI) into highly aggressive tumor cells, even acting on cell plasticity. Furthermore, in oral metastatic cell line (HN12), SK2 contributed even more to the tumorigenesis, inducing proliferation and tumor growth. Our work reveals the intriguing role of SK2 as an oral tumor promoter and regulator of different pathways and cellular processes.

Topics: Autophagy; Carcinogenesis; Carcinoma, Squamous Cell; Cell Proliferation; Cell Transformation, Neoplastic; Head and Neck Neoplasms; Humans; Keratinocytes; Mouth Neoplasms; Squamous Cell Carcinoma of Head and Neck

Despite progress in the understanding of the biology and genetics of melanoma, no effective treatment against this cancer is available. The adjacent microenvironment has an important role in melanoma progression. Defining the molecular signals that control the bidirectional dialog between malignant cells and the surrounding stroma is crucial for efficient targeted therapy. Our study aimed at defining the role of sphingosine-1-phosphate (S1P) in melanoma-stroma interactions. Transcriptomic analysis of human melanoma cell lines showed increased expression of sphingosine kinase-1 (SPHK1), the enzyme that produces S1P, as compared with normal melanocytes. Such an increase was also observed by immunohistochemistry in melanoma specimens as compared with nevi, and occurred downstream of ERK activation because of BRAF or NRAS mutations. Importantly, migration of melanoma cells was not affected by changes in SPHK1 activity in tumor cells, but was stimulated by comparable modifications of S1P-metabolizing enzymes in cocultured dermal fibroblasts. Reciprocally, incubation of fibroblasts with the conditioned medium from SPHK1-expressing melanoma cells resulted in their differentiation to myofibroblasts, increased production of matrix metalloproteinases and enhanced SPHK1 expression and activity. In vivo tumorigenesis experiments showed that the lack of S1P in the microenvironment prevented the development of orthotopically injected melanoma cells. Finally, local tumor growth and dissemination were enhanced more efficiently by coinjection of wild-type skin fibroblasts than by fibroblasts from Sphk1(-/-) mice. This report is the first to document that SPHK1/S1P modulates the communication between melanoma cells and dermal fibroblasts. Altogether, our findings highlight SPHK1 as a potential therapeutic target in melanoma progression.

Topics: Aldehyde-Lyases; Animals; Cell Communication; Cell Differentiation; Cell Movement; Cell Transformation, Neoplastic; Extracellular Signal-Regulated MAP Kinases; Female; Fibroblasts; Gene Expression Profiling; GTP Phosphohydrolases; HEK293 Cells; Humans; Lysophospholipids; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Melanoma; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Myofibroblasts; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins B-raf; RNA Interference; RNA, Messenger; RNA, Small Interfering; Skin Neoplasms; Sphingosine

Sphingosine kinase 1 (SK1) is a lipid kinase that catalyses the formation of sphingosine-1-phosphate (S1P). Considerable evidence has implicated elevated cellular SK1 in tumour development, progression and disease severity. In particular, SK1 has been shown to enhance cell survival and proliferation and induce neoplastic transformation. Although S1P has been found to have both cell-surface G-protein-coupled receptors and intracellular targets, the specific downstream pathways mediating oncogenic signalling by SK1 remain poorly defined. Here, using a gene expression array approach, we have demonstrated a novel mechanism whereby SK1 regulates cell survival, proliferation and neoplastic transformation through enhancing expression of transferrin receptor 1 (TFR1). We showed that elevated levels of SK1 enhanced total as well as cell-surface TFR1 expression, resulting in increased transferrin uptake into cells. Notably, we also found that SK1 activation and localization to the plasma membrane, which are critical for its oncogenic effects, are necessary for regulation of TFR1 expression specifically through engagement of the S1P G-protein coupled receptor, S1P2. Furthermore, we showed that blocking TFR1 function with a neutralizing antibody inhibits SK1-induced cell proliferation, survival and neoplastic transformation of NIH3T3 fibroblasts. Similar effects were observed following antagonism of S1P2. Together these findings suggest that TFR1 has an important role in SK1-mediated oncogenesis.

Topics: Animals; Antigens, CD; Cell Line; Cell Transformation, Neoplastic; Fluorescent Antibody Technique; Gene Expression Regulation; Gene Knockdown Techniques; Humans; Immunoblotting; Mice; Neoplasms; Oligonucleotide Array Sequence Analysis; Phosphotransferases (Alcohol Group Acceptor); Real-Time Polymerase Chain Reaction; Receptors, Transferrin; RNA, Small Interfering; Signal Transduction; Transfection

Inflammatory bowel disease is an important risk factor for colorectal cancer. We show that sphingosine-1-phosphate (S1P) produced by upregulation of sphingosine kinase 1 (SphK1) links chronic intestinal inflammation to colitis-associated cancer (CAC) and both are exacerbated by deletion of Sphk2. S1P is essential for production of the multifunctional NF-κB-regulated cytokine IL-6, persistent activation of the transcription factor STAT3, and consequent upregulation of the S1P receptor, S1PR1. The prodrug FTY720 decreased SphK1 and S1PR1 expression and eliminated the NF-κB/IL-6/STAT3 amplification cascade and development of CAC, even in Sphk2(-/-) mice, and may be useful in treating colon cancer in individuals with ulcerative colitis. Thus, the SphK1/S1P/S1PR1 axis is at the nexus between NF-κB and STAT3 and connects chronic inflammation and CAC.

Topics: Animals; Cell Transformation, Neoplastic; Colitis; Colon; Fingolimod Hydrochloride; Gene Deletion; Gene Expression Regulation, Neoplastic; Interleukin-6; Lysophospholipids; Mice; NF-kappa B; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Sphingosine; STAT3 Transcription Factor; Tumor Microenvironment

p53 is a crucial tumor suppressor that is mutated or deleted in a majority of cancers. Exactly how p53 prevents tumor progression has proved elusive for many years; however, this information is crucial to define targets for chemotherapeutic development that can effectively restore p53 function. Bioactive sphingolipids have recently emerged as important regulators of proliferative, apoptotic and senescent cellular processes. In this study, we demonstrate that the enzyme sphingosine kinase 1 (SK1), a critical enzyme in the regulation of the key bioactive sphingolipids ceramide, sphingosine and sphingosine-1-phosphate (S1P), serves as a key downstream target for p53 action. Our results show that SK1 is proteolysed in response to genotoxic stress in a p53-dependent manner. p53 null mice display elevation of SK1 levels and a tumor-promoting dysregulation of bioactive sphingolipids in which the anti-growth sphingolipid ceramide is decreased and the pro-growth sphingolipid S1P is increased. Importantly, deletion of SK1 in p53 null mice completely abrogated thymic lymphomas in these mice and prolonged their life span by ~30%. Deletion of SK1 also significantly attenuated the formation of other cancers in p53 heterozygote mice. The mechanism of p53 tumor suppression by loss of SK1 is mediated by elevations of sphingosine and ceramide, which in turn were accompanied by increased expression of cell cycle inhibitors and tumor cell senescence. Thus, targeting SK1 may restore sphingolipid homeostasis in p53-dependent tumors and provide insights into novel therapeutic approaches to cancer.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cellular Senescence; Enzyme Activation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Tumor Burden; Tumor Suppressor Protein p53

SPHK1 expression is elevated in gastric cancer and is associated with shorter survival times for patients. However, the molecular mechanism of SPHK1 up-regulation in gastric cancer remains unclear. In the present study, we report that miR-124 down-regulated SPHK1 expression by directly targeting its 3'-untranslated region (3'-UTR) and that miR-124 expression was inversely correlated with SPHK1 expression in gastric cancer samples. Furthermore, we demonstrated that, similar to the effect of silencing SPHK1, up-regulation of miR-124 markedly inhibited proliferation and tumourigenicity of gastric cancer cells both in vitro and in vivo. This was found to be mechanistically associated with induction of cyclin-dependent kinase inhibitors p21$^{{\\rm Cip1}}$ and p27$^{{\\rm Kip1}}$, enhancement of the transcriptional activity of FOXO1 and suppression of AKT activity. Moreover, we showed that the re-introduction of SPHK1 (without the 3'-UTR), but not with the 3'-UTR, could abrogate the miR-124-mediated induction of p21$^{{\\rm Cip1}}$ and p27$^{{\\rm Kip1}}$, as well as rescue the miR-124-induced proliferation inhibition. Together, these results suggest that miR-124 has an important role in the suppression of gastric cancer and presents a novel mechanism of miRNA-mediated SPHK1 expression in cancer cells.

Topics: Adenocarcinoma; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Down-Regulation; Forkhead Box Protein O1; Forkhead Transcription Factors; Humans; In Vitro Techniques; MicroRNAs; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-akt; Signal Transduction; Stomach Neoplasms

Sphingosine kinase 1 (SK1) is an important enzyme involved in the production of the bioactive lipid sphingosine 1-phosphate (S1P). SK1 is overexpressed in many forms of cancer, however, the contribution of SK1 to cancer progression is still unclear. One of the best characterized mutations found in several forms of human cancer is an activating point mutation in the Ras oncogene, which disrupts its GTPase activity and leads to stimulation of the MEK/ERK pathway. Because SK1 activity and subcellular localization have been shown to be regulated by ERK, we wished to investigate the effect of oncogenic Ras, a potent activator of the Raf/MEK/ERK pathway, on the activity of SK1 and sphingolipid metabolism. Using HEK293T cells transiently transfected with the K-RasG12V oncogene and both wild type and Sphk1(-/-) mouse embryonic fibroblasts stably infected with retroviral K-RasG12V, we found that K-RasG12V increases the production of S1P and decreases the production of ceramide in a SK1-dependent manner. In addition, we found that expression of the K-RasG12V oncogene leads to plasma membrane localization of SK1 and a reduction in cytosolic levels of SK1. This effect is likely mediated by the Raf/MEK/ERK pathway as constitutively active B-Raf or MEK1 are able to activate SK1, but constitutively active Akt1 is not. We believe this research has important implications for how sphingolipids may be contributing to oncogenic transformation and provide some of the first evidence for oncogenes inducing specific changes in sphingolipid metabolism through SK1 regulation.

Topics: Animals; Cell Membrane; Cell Transformation, Neoplastic; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; HEK293 Cells; Humans; Mice; Mice, Inbred C57BL; Mice, Transgenic; Models, Biological; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; Sphingolipids; Subcellular Fractions

Sphingosine kinase 1 (SK1) catalyses the formation of bioactive phospholipid sphingosine 1-phosphate (S1P). Elevated cellular SK1 activity and S1P levels enhance cell proliferation and survival, and are strongly implicated in tumourigenesis. Regulation of SK1 activity can occur through various mechanisms, including phosphorylation and protein-protein interactions. We have previously shown that eukaryotic elongation factor 1A (eEF1A) interacts with and directly activates SK1, but the mechanisms regulating this were undefined. Notably, eEF1A has GTPase activity and can exist in GTP- or GDP-bound forms, which are associated with distinct structural conformations of the protein. Here, we show that the guanine nucleotide-bound state of eEF1A regulates its ability to activate SK1, with eEF1A.GDP, but not eEF1A.GTP, enhancing SK1 activity in vitro. Furthermore, we show that enhancing cellular eEF1A.GDP levels through expression of a guanine nucleotide dissociation inhibitor of eEF1A, translationally controlled tumour protein (TCTP), increased SK1 activity in cells. We also examined a truncated isoform of eEF1A1, termed prostate tumour inducer-1 (PTI-1), which can induce neoplastic cell transformation through undefined mechanisms. PTI-1 lacks the G protein domain of eEF1A1 and is therefore unable to undergo the GTP-binding-induced conformational change. Notably, we found that PTI-1 can directly activate SK1 and that this seems to be essential for neoplastic transformation induced by PTI-1, as chemical SK1 inhibitors or overexpression of a dominant-negative SK1 blocked this process. Thus, this study defines the mechanism regulating eEF1A-mediated SK1 activation, and also establishes SK1 as being integral for PTI-1-induced oncogenesis.

Topics: Animals; Base Sequence; Cell Transformation, Neoplastic; CHO Cells; Cricetinae; Cricetulus; DNA Primers; Enzyme Activation; Guanine Nucleotides; Humans; Peptide Elongation Factor 1; Phosphotransferases (Alcohol Group Acceptor); Tumor Protein, Translationally-Controlled 1

Sphingosine kinase 1 (SK1) is an important regulator of cellular signalling that has gained recent attention as a potential target for anti-cancer therapies. SK1 activity, subcellular localization and oncogenic function are regulated by phosphorylation and dephosphorylation at Ser225. ERK1/2 have been identified as the protein kinases responsible for phosphorylation and activation of SK1. Conversely, dephosphorylation and deactivation of SK1 occurs by protein phosphatase 2A (PP2A). Active PP2A, however, is a heterotrimer, composed of tightly associated catalytic and structural subunits that can interact with an array of regulatory subunits, which are critical for determining holoenzyme substrate specificity and subcellular localization. Thus, PP2A represents a large family of holoenzyme complexes with different activities and diverse substrate specificities. To date the regulatory subunit essential for targeting PP2A to SK1 has remained undefined. Here, we demonstrate a critical role for the B'α (B56α/PR61α/PPP2R5A) regulatory subunit of PP2A in SK1 dephosphorylation. B'α was found to interact with the c-terminus of SK1, and reduce SK1 phosphorylation when overexpressed, while having no effect on upstream ERK1/2 activation. siRNA-mediated knockdown of B'α increased SK1 phosphorylation, activity and membrane localization of endogenous SK1. Furthermore, overexpression of B'α blocked agonist-induced translocation of SK1 to the plasma membrane and abrogated SK1-induced neoplastic transformation of NIH3T3 fibroblasts. Thus, the PP2A-B'α holoenzyme appears to function as an important endogenous regulator of SK1.

Topics: Animals; Cell Membrane; Cell Transformation, Neoplastic; Gene Knockdown Techniques; HEK293 Cells; Humans; Immunoprecipitation; Mice; NIH 3T3 Cells; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Binding; Protein Phosphatase 2; Protein Transport

FGF1, a widely expressed proangiogenic factor involved in tissue repair and carcinogenesis, is released from cells through a non-classical pathway independent of endoplasmic reticulum and Golgi. Although several proteins participating in FGF1 export were identified, genetic mechanisms regulating this process remained obscure. We found that FGF1 export and expression are regulated through Notch signaling mediated by transcription factor CBF1 and its partner MAML. The expression of a dominant negative (dn) form of CBF1 in 3T3 cells induces transcription of FGF1 and sphingosine kinase 1 (SphK1), which is a component of FGF1 export pathway. dnCBF1 expression stimulates the stress-independent release of transduced FGF1 from NIH 3T3 cells and endogenous FGF1 from A375 melanoma cells. NIH 3T3 cells transfected with dnCBF1 form colonies in soft agar and produce rapidly growing highly angiogenic tumors in nude mice. The transformed phenotype of dnCBF1 transfected cells is efficiently blocked by dn forms of FGF receptor 1 and S100A13, which is a component of FGF1 export pathway. FGF1 export and acceleration of cell growth induced by dnCBF1 depend on SphK1. Similar to dnCBF1, dnMAML transfection induces FGF1 expression and release, and accelerates cell proliferation. The latter effect is strongly decreased in FGF1 null cells. We suggest that the regulation of FGF1 expression and release by CBF1-mediated Notch signaling can play an important role in tumor formation.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Fibroblast Growth Factor 1; Humans; Immunoglobulin J Recombination Signal Sequence-Binding Protein; Melanoma; Mice; Mice, Nude; Neovascularization, Pathologic; NIH 3T3 Cells; Nuclear Proteins; Phosphotransferases (Alcohol Group Acceptor); Receptors, Notch; S100 Proteins; Signal Transduction; Transcription Factors; Transfection

Sphingosine kinase 1 (SphK1) phosphorylates sphingosine to form sphingosine-1-phosphate (S1P) and is a critical regulator of sphingolipid-mediated functions. Cell-based studies suggest a tumor-promoting function for the SphK1/S1P pathway. Also, our previous studies implicated the SphK1/S1P pathway in the induction of the arachidonic acid cascade, a major inflammatory pathway involved in colon carcinogenesis. Therefore, we investigated whether the SphK1/S1P pathway is necessary for mediating carcinogenesis in vivo. Here, we report that 89% (42/47) of human colon cancer samples stained positively for SphK1, whereas normal colon mucosa had negative or weak staining. Adenomas had higher expression of SphK1 vs. normal mucosa, and colon cancers with metastasis had higher expression of SphK1 than those without metastasis. In the azoxymethane (AOM) murine model of colon cancer, SphK1 and S1P were significantly elevated in colon cancer tissues compared to normal mucosa. Moreover, blood levels of S1P were higher in mice with colon cancers than in those without cancers. Notably, SphK1(-/-) mice subjected to AOM had significantly less aberrant crypt foci (ACF) formation and significantly reduced colon cancer development. These results are the first in vivo evidence that the SphK1/S1P pathway contributes to colon carcinogenesis and that inhibition of this pathway is a potential target for chemoprevention.

Topics: Animals; Azoxymethane; Cell Transformation, Neoplastic; Colonic Neoplasms; Cyclooxygenase 2; Dextran Sulfate; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Inflammation; Lysophospholipids; Mice; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

The mitogenic role of sphingosine-1-phosphate (S1P) and its involvement in basic fibroblast growth factor (bFGF)-induced proliferation were examined in primary cultures of cerebellar astrocytes. Exposure to bFGF resulted in a rapid increase of extracellular S1P formation, bFGF inducing astrocytes to release S1P, but not sphingosine kinase, in the extracellular milieu. The SK inhibitor N,N-dimethylsphingosine inhibited S1P release as well as bFGF-induced growth stimulation. S1P application in quiescent astrocytes caused a dose-dependent increase in DNA synthesis. This gliotrophic effect was induced by a brief exposure to low nanomolar S1P, mimicked by the S1P receptor agonist dihydro-S1P, and inhibited by pertussis toxin (PTX), an inactivator of G(i)/G(o)-proteins. S1P also induced activation of extracellular signal-regulated kinase that was inhibited again by PTX. Moreover, the S1P lyase inhibitor 4-deoxypyridoxine induced the cellular accumulation of S1P but did not affect DNA synthesis. These results support the view that S1P exerted a mitogenic effect on cerebellar astrocytes extracellularly, most likely through cell surface S1P receptors. In agreement, mRNAs for S1P1, S1P2, and S1P3 receptors are expressed in cerebellar astrocytes (Anelli et al., 2005. J Neurochem 92:1204-1215). Ceramide, a negative regulator of astrocyte proliferation and down-regulated by bFGF (Riboni et al., 2002. Cerebellum 1:129-135), efficiently inhibited S1P-induced proliferation. The S1P action appears to be part of an autocrine/paracrine cascade stimulated by bFGF and, together with ceramide down-regulation, essential for astrocytes to respond to bFGF. The results suggest that S1P and bFGF/S1P may play an important role in physiopathological glial proliferation, such as brain development, reactive gliosis and brain tumor formation.

Topics: Animals; Animals, Newborn; Astrocytes; Astrocytoma; Brain Neoplasms; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Ceramides; Cerebellum; DNA Replication; Enzyme Inhibitors; Extracellular Fluid; Extracellular Signal-Regulated MAP Kinases; Fibroblast Growth Factor 2; Gliosis; GTP-Binding Protein alpha Subunits, Gi-Go; Lysophospholipids; Mitosis; Phosphotransferases (Alcohol Group Acceptor); Rats; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

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.

Topics: Apoptosis; Cell Membrane; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; DNA Primers; Fluorescent Antibody Technique; Humans; Mutation; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Polymerase Chain Reaction; Protein Transport; Signal Transduction; Transfection

The erythroleukemia developed by spi-1/PU.1-transgenic mice is a model of multistage oncogenic process. Isolation of tumor cells representing discrete stages of leukemic progression enables the dissection of some of the critical events required for malignant transformation. To elucidate the molecular mechanisms of multistage leukemogenesis, we developed a microarray transcriptome analysis of nontumorigenic (HS1) and tumorigenic (HS2) proerythroblasts from spi-1-transgenic mice. The data show that transcriptional up-regulation of the sphingosine kinase gene (SPHK1) is a recurrent event associated with the tumorigenic phenotype of these transgenic proerythroblasts. SPHK1 is an enzyme of the metabolism of sphingolipids, which are essential in several biologic processes, including cell proliferation and apoptosis. HS1 erythroleukemic cells engineered to overexpress the SPHK1 protein exhibited growth proliferative advantage, increased clonogenicity, and resistance to apoptosis in reduced serum level by a mechanism involving activation of the extracellular signal-related kinases 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways. In addition, SPHK1-overexpressing HS1 cells acquired tumorigenicity when engrafted in vivo. Finally, enforced expression of a dominant-negative mutant of SPHK1 in HS2 tumorigenic cells or treatment with a pharmacologic inhibitor reduced both cell growth and apoptosis resistance. Altogether, these data suggest that overexpression of the sphingosine kinase may represent an oncogenic event during the multistep progression of an erythroleukemia.

Topics: Animals; Cell Line; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Cloning, Molecular; Disease Progression; Erythroblasts; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Genes, Dominant; Leukemia, Erythroblastic, Acute; Mice; Mice, Transgenic; Neoplasm Transplantation; Neoplasms, Experimental; Phosphotransferases (Alcohol Group Acceptor); Protein Isoforms; Up-Regulation

Sphingosine kinase (SphK) is a highly conserved lipid kinase that phosphorylates sphingosine to form sphingosine-1-phosphate (S1P). S1P/SphK has been implicated as a signalling pathway to regulate diverse cellular functions [1-3], including cell growth, proliferation and survival [4-8]. We report that cells overexpressing SphK have increased enzymatic activity and acquire the transformed phenotype, as determined by focus formation, colony growth in soft agar and the ability to form tumours in NOD/SCID mice. This is the first demonstration that a wild-type lipid kinase gene acts as an oncogene. Using a chemical inhibitor of SphK, or an SphK mutant that inhibits enzyme activation, we found that SphK activity is involved in oncogenic H-Ras-mediated transformation, suggesting a novel signalling pathway for Ras activation. The findings not only point to a new signalling pathway in transformation but also to the potential of SphK inhibitors in cancer therapy.

Topics: 3T3 Cells; Animals; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Genes, ras; Humans; Lysophospholipids; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasms, Experimental; Oncogenes; Phosphotransferases (Alcohol Group Acceptor); ras Proteins; Signal Transduction; Sphingosine; Transfection