sphingosine-kinase and Adenocarcinoma

LncRNA MEG3 is involved in the pathogenesis of several types of cancers. While its participation and function network in colorectal (CR) adenocarcinoma, which is one of the most common malignancies, still hasn't been well studied. Therefore, our study aimed to investigate the role of MEG3 in colorectal adenocarcinoma and to explore the possibly related mechanisms.. Tumor tissues and adjacent healthy tissues were collected from colorectal adenocarcinoma patients. Blood samples were collected from both colorectal adenocarcinoma patients and healthy controls to prepare serum sample. Expression of MEG3 in those tissues was detected by qRT-PCR. MEG3 knockdown and sphingosine kinase 1 (SPHK1) overexpression colorectal adenocarcinoma cell lines were established. Its effects on cell proliferation and apoptosis were investigated by CCK-8 assay and MTT assay, respectively. Effects of MEG3 overexpression on TGF-β1 and SPHK1 were investigated by Western blot.. MEG3 expression level was decreased in tumor tissues than that in adjacent healthy tissues. Serum level of MEG3 was lower in cancer patients than that in healthy controls, and the serum level decreased with the increased stage of primary tumor. Serum TGF-β1 can be used to predict colorectal adenocarcinoma and its prognosis accurately. MEG3 knockdown and SPHK1 overexpression promoted tumor cell proliferation, but inhibited cell apoptosis. MEG3 knockdown also increased the expression level of TGF-β1 and SPHK1. Treatment with TGF-β1 inhibitor reduced the expression level of SPHK1 but showed no significant effects on MEG3. SPHK1 overexpression showed no significant effects on MEG3 and TGF-β1 expression.. Downregulation of lncRNA MEG3 can promote colorectal adenocarcinoma cell proliferation and inhibit the apoptosis by up-regulating TGF-β1 and its downstream sphingosine kinase 1.

Topics: Adenocarcinoma; Adult; Aged; Apoptosis; Case-Control Studies; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Down-Regulation; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Phosphotransferases (Alcohol Group Acceptor); RNA, Long Noncoding; Signal Transduction; Transforming Growth Factor beta1; Young Adult

Strong experimental evidence in animal and cellular models supports a pivotal role of sphingosine kinase-1 (SK1) in oncogenesis. In many human cancers, SK1 levels are upregulated and these increases are linked to poor prognosis in patients. Here, by employing untargeted NMR-based metabolomic profiling combined with functional validations, we report the crucial role of SK1 in the metabolic shift known as the Warburg effect in A2780 ovarian cancer cells. Indeed, expression of SK1 induced a high glycolytic rate, characterized by increased levels of lactate along with increased expression of the proton/monocarboxylate symporter MCT1, and decreased oxidative metabolism, associated with the accumulation of intermediates of the tricarboxylic acid cycle and reduction in CO

Topics: Adenocarcinoma; Animals; Carbon Dioxide; Cell Cycle Proteins; Cell Line, Tumor; Female; Glucose; Glucose Transporter Type 3; Glycolysis; Humans; Lactic Acid; Magnetic Resonance Imaging; Metabolomics; Mitochondria; Oncogene Proteins; Ovarian Neoplasms; Oxidation-Reduction; Phosphotransferases (Alcohol Group Acceptor); Prognosis; Tricarboxylic Acids; Up-Regulation

To examine the expression of SphK1, an oncogenic kinase that produces sphingosine 1-phosphate (S1P), and its correlation with the expression of LPAR2, a major lysophosphatidic acid (LPA) receptor overexpressed in various cancers, in human colorectal cancer.. Real-time reverse-transcription polymerase chain reaction was used to measure the mRNA expression of SphK1, LPAR2, and the three major S1P receptors in 27 colorectal cancer samples and corresponding normal tissue samples. We also examined the correlation between the expression of SphK1 and LPAR2.. Colorectal cancer tissue in 22 of 27 patients had higher levels of SphK1 mRNA than in normal tissue. In two-thirds of the samples, SphK1 mRNA expression was more than two-fold higher than in normal tissue. Consistent with previous reports, LPAR2 mRNA expression in 20 of 27 colorectal cancer tissue samples was higher compared to normal tissue samples. Expression profiles of all three major S1P receptors, S1PR1, S1PR2, and S1PR3, varied without any trend, with no significant difference in expression between cancer and normal tissues. A highly significant positive correlation was found between SphK1 and LPAR2 expression [Pearson's correlation coefficient (r) = 0.784 and P < 0.01]. The mRNA levels of SphK1 and LPAR2 did not correlate with TNM stage.. Our findings suggest that S1P and LPA may play important roles in the development of colorectal cancer via the upregulation of SphK1 and LPAR2, both of which could serve as new therapeutic targets in the treatment of colorectal cancer.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Colorectal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Neoplasm Staging; Phosphotransferases (Alcohol Group Acceptor); Real-Time Polymerase Chain Reaction; Receptors, Lysophosphatidic Acid; Receptors, Lysosphingolipid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sphingosine-1-Phosphate Receptors; Up-Regulation

There are no effective treatments for pancreatic cancer peritoneal carcinomatosis (PC) or cancer dissemination in abdominal cavity. Sphingosine-1-phosphate (S1P), a bioactive lipid mediator produced by sphingosine kinases (SphK1 and SphK2), plays critical roles in cancer progression. We reported that SphK1, but not SphK2, is responsible for S1P export from breast cancer cells and recently discovered that S1P is linked to inflammation and cancer in colitis-associated cancer progression. Given the fact that inflammation is known to be essential for the establishment and progression of PC, we hypothesized that SphK1 in the host animals is involved in progression of pancreatic cancer PC.. Murine pancreatic adenocarcinoma panc02-luc cells were intraperitoneally injected into wildtype or SphK1 knockout (KO) mice to generate a syngeneic PC model. Cell proliferation and apoptosis were determined by Ki67 and TUNEL staining, respectively.. All the animals developed panc02-luc PC. SphK1 KO mice developed significantly less tumor burden, less total tumor weight, and fewer number of PC nodules at 14 d after implantation. Histologically, less inflammatory cell infiltration and less cancer cell proliferation were observed in the tumors. There was no difference in apoptosis.. Our results raise an intriguing possibility that S1P generated by SphK1 in the host promotes pancreatic cancer PC progression by stimulation of proliferation of cancer cells.

Topics: Adenocarcinoma; Animals; Apoptosis; Biomarkers, Tumor; Cell Proliferation; In Situ Nick-End Labeling; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neoplasm Transplantation; Pancreatic Neoplasms; Peritoneal Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Tumor Burden

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.

Topics: Adenocarcinoma; Adenosine Triphosphate; Animals; Antineoplastic Agents; Apoptosis; Binding Sites; Cell Line; Cell Line, Tumor; Enzyme Inhibitors; Female; HEK293 Cells; Humans; Lung Neoplasms; Male; MCF-7 Cells; Mice; Mice, Transgenic; Molecular Conformation; Mutagenesis; Mutation; Neoplasm Transplantation; Neovascularization, Pathologic; Phosphotransferases (Alcohol Group Acceptor); Protein Binding; Sphingolipids

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

The pro-apoptotic lipid sphingosine is phosphorylated by sphingosine kinases 1 and 2 (SK1 and SK2) to generate the mitogenic lipid sphingosine-1-phosphate (S1P). We previously reported that inhibition of SK activity delays tumor growth in a mouse mammary adenocarcinoma model. Because SK inhibitors and the multikinase inhibitor sorafenib both suppress the MAP kinase pathway, we hypothesized that their combination may provide enhanced inhibition of tumor growth. Therefore, we evaluated the effects of two SK inhibitors, ABC294640 (a SK2-specific inhibitor) and ABC294735 (a dual SK1/SK2 inhibitor), alone and in combination with sorafenib on human pancreatic adenocarcinoma (Bxpc-3) and kidney carcinoma (A-498) cells in vitro and in vivo. Exposure of either Bxpc-3 or A-498 cells to combinations of ABC294640 and sorafenib or ABC294735 and sorafenib resulted in synergistic cytotoxicity, associated with activation of caspases 3/7 and DNA fragmentation. Additionally, strong decreases in ERK phosphorylation were observed in Bxpc-3 and A-498 cells exposed to either the sorafenib/ABC294640 or the sorafenib/ABC294735 combination. Oral administration of either ABC294640 or ABC294735 to mice led to a delay in tumor growth in both xenograft models without overt toxicity to the animals. Tumor growth delay was potentiated by co-administration of sorafenib. These studies show that combination of an SK inhibitor with sorafenib causes synergistic inhibition of cell growth in vitro, and potentiates antitumor activity in vivo. Thus, a foundation is established for clinical trials evaluating the efficacy of combining these signaling inhibitors.

Topics: Adamantane; Adenocarcinoma; Administration, Oral; Animals; Antineoplastic Combined Chemotherapy Protocols; Benzenesulfonates; Caspase 3; Caspase 7; Catechols; Cell Line, Tumor; DNA Fragmentation; Drug Synergism; Female; Humans; Kidney Neoplasms; Mice; Mice, SCID; Niacinamide; Pancreatic Neoplasms; Phenylurea Compounds; Phosphotransferases (Alcohol Group Acceptor); Pyridines; Sorafenib; Xenograft Model Antitumor Assays

Sphingosine kinases (SK) catalyze the formation of sphingosine-1-phosphate (S1P) which plays a crucial role in cell growth and survival. Here, we show that prolactin (PRL) biphasically activates the SK-1, but not the SK-2 subtype, in the breast adenocarcinoma cell-line MCF7. A first peak occurs after minutes of stimulation and is followed by a second delayed activation after hours of stimulation. A similar biphasic effect on SK-1 activity is seen for 17beta-estradiol (E(2)). The delayed activation of SK-1 derives from an upregulated mRNA and protein expression and is due to increased SK-1 promoter activity and mechanistically involves STAT5 activation as well as protein kinase C and the classical mitogen-activated protein kinases. Furthermore, glucocorticoids also block both hormone-induced SK-1 expression and activity. Functionally, long-term stimulation of MCF7 cells with PRL or E(2) is well known to trigger increased cell proliferation and migration. Both hormone-induced cell responses critically involve SK-1 activation since the depletion of SK-1, but not SK-2, by siRNA transfection abolishes the hormone-induced cell proliferation and migration. In summary, our data show that PRL and E(2) cause a pronounced delayed SK-1 activation which is due to increased gene transcription, and critically determines the capability of cells to grow and move. Thus, the SK-1 may represent a novel attractive target for anti-tumor therapy.

Topics: Adenocarcinoma; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; Estradiol; Humans; Phosphotransferases (Alcohol Group Acceptor); Prolactin; Promoter Regions, Genetic; RNA, Small Interfering; Up-Regulation

Elevated BCL-2 is one mechanism of therapeutic resistance in prostate cancer (PC), and new approaches are needed to overcome such resistance.. We evaluated the effects of BCL-2 over-expression in human prostatic adenocarcinoma cells on their susceptibility to sphingolipids (SLs) and to the sphingosine kinase (SpK) inhibitor, SKI II.. In survival assays, no significant differences were observed in the responses to sphingosine or ceramide among parental PC-3 cells lacking detectable BCL-2 and BCL-2 over-expressing PC-3 transfectants; similarly, the responses to dimethyl-sphingosine (DMSP) of parental LNCaP cells and a BCL-2 over-expressing LNCaP transfectant were equivalent. SKI II induced protracted, BCL-2-independent survival loss in both PC-3 and LNCaP parental/transfectant pairs; in contrast, DMSP induced rapid cell shrinkage, caspase activation and caspase-dependent DNA fragmentation. DMSP-induced DNA fragmentation and loss of mitochondrial membrane potential were equivalent in BCL-2 transfectants and parental PC-3 cells and were not associated with BCL-2 downregulation. DMSP-mediated cytotoxicity was not associated with the enhanced production of reactive oxygen intermediates. SL analyses of parental and transfectant PC-3 cells did not reveal increased levels of sphingosine-1-phosphate in the BCL-2 transfectants; further, there only a modest early shift, corresponding to apoptotic onset, in pro- versus anti-apoptotic SLs in response to DMSP treatment.. Thus, in contrast to the inhibitory effects of BCL-2 on apoptosis induced by various agents in tumor cells, SKI II and selected pro-apoptotic SLs appear atypical in their independence from such inhibition, and may have merits as new candidates for treatment of AI PC.

Topics: Adenocarcinoma; Apoptosis; Cell Line, Tumor; Cell Survival; Ceramides; Down-Regulation; Enzyme Inhibitors; Humans; Male; Phosphotransferases (Alcohol Group Acceptor); Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Sphingosine; Thiazoles

Activation of sphingosine kinase-1 (SK1) by overexpression or agonist stimulation promotes cell proliferation, survival, and anti-apoptosis. Studies on the function of endogenous SK1 are lacking. Endogenous SK1 has been shown to be down-regulated under stress, and knockdown of the enzyme reduces the percentage of viable MCF-7 breast cancer cells (Taha, T. A. et al. 2004. J. Biol. Chem. 279, 20546-20554). In this study, we examined the mechanisms by which SK1 loss affects the growth of cells. Knockdown of the enzyme by small interfering RNA caused cell cycle arrest and induced apoptosis. Cell death involved effector caspase activation, cytochrome c release and Bax oligomerization in the mitochondrial membrane, thus placing SK1 knockdown upstream of the mitochondrial pathway of apoptosis. SK1 knockdown also induced significant increases in ceramide levels in whole cells and in mitochondria enriched fractions of cells. Inhibition of de novo sphingolipid biosynthesis with myriocin significantly attenuated Bax oligomerization and downstream caspase activation after SK1 loss. These studies for the first time implicate endogenous SK1 as an important survival enzyme in MCF-7 cells and link the biological consequences of knocking down the enzyme to its biochemical role as a regulator of sphingolipid metabolism.

Topics: Adenocarcinoma; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Breast Neoplasms; Caspases; Cell Cycle; Cell Line, Tumor; Ceramides; Cytochromes c; Enzyme Activation; Fatty Acids, Monounsaturated; Female; Gene Targeting; Humans; Mitochondria; Neoplasm Proteins; Phosphotransferases (Alcohol Group Acceptor); RNA, Messenger; RNA, Small Interfering; Sphingolipids

Sphingosine kinase 1 (SK1) is a key enzyme critical to the sphingolipid metabolic pathway responsible for catalyzing the formation of the bioactive lipid sphingosine-1-phosphate. SK1-mediated production of sphingosine-1-phosphate has been shown to stimulate such biological processes as cell growth, differentiation, migration, angiogenesis, and inhibition of apoptosis. In this study, cell type-specific immunolocalization of SK1 was examined in the bronchus/terminal bronchiole of the lung. Strong immunopositive staining was evident at the apical surface of pseudostratified epithelial cells of the bronchus and underlying smooth muscle cells, submucosal serous glands, immature chondrocytes, type II alveolar cells, foamy macrophages, endothelial cells of blood vessels, and neural bundles. Immunohistochemical screening for SK1 expression was performed in 25 samples of normal/tumor patient matched non-small-cell lung cancer tissue and found that 25 of 25 tumor samples (carcinoid [5 samples], squamous [10 samples], and adenocarcinoma tumors [10 samples]), exhibited overwhelmingly positive immunostaining for SK1 as compared with patient-matched normal tissue. In addition, an approximately 2-fold elevation of SK1 mRNA expression was observed in lung cancer tissue versus normal tissue, as well as in several other solid tumors. Taken together, these findings define the localization of SK1 in lung and provide clues as to how SK1 may play a role in normal lung physiology and the pathophysiology of lung cancer.

Topics: Adenocarcinoma; Animals; Antibody Specificity; Bronchi; Carcinoid Tumor; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Humans; Immunohistochemistry; Lung; Lung Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Rabbits; RNA, Messenger

Systemic chemotherapy was considered of modest efficacy in prostate cancer until the recent introduction of taxanes. We took advantage of the known differential effect of camptothecin and docetaxel on human PC-3 and LNCaP prostate cancer cells to determine their effect on sphingosine kinase-1 (SphK1) activity and subsequent ceramide/sphingosine 1-phosphate (S1P) balance in relation with cell survival. In vitro, docetaxel and camptothecin induced strong inhibition of SphK1 and elevation of the ceramide/S1P ratio only in cell lines sensitive to these drugs. SphK1 overexpression in both cell lines impaired the efficacy of chemotherapy by decreasing the ceramide/S1P ratio. Alternatively, silencing SphK1 by RNA interference or pharmacologic inhibition induced apoptosis coupled with ceramide elevation and loss of S1P. The differential effect of both chemotherapeutics was confirmed in an orthotopic PC-3/green fluorescent protein model established in nude mice. Docetaxel induced a stronger SphK1 inhibition and ceramide/S1P ratio elevation than camptothecin. This was accompanied by a smaller tumor volume and the reduced occurrence and number of metastases. SphK1-overexpressing PC-3 cells implanted in animals developed remarkably larger tumors and resistance to docetaxel treatment. These results provide the first in vivo demonstration of SphK1 as a sensor of chemotherapy.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Camptothecin; Ceramides; Disease Models, Animal; Docetaxel; Flow Cytometry; Green Fluorescent Proteins; Humans; Lysophospholipids; Male; Mice; Mice, Nude; Microscopy, Fluorescence; Neoplasm Recurrence, Local; Neoplasms, Hormone-Dependent; Phosphotransferases (Alcohol Group Acceptor); Prostatic Neoplasms; RNA Interference; Sphingosine; Taxoids; Tumor Cells, Cultured

The sphingolipid metabolite, sphingosine-1-phosphate (S1P), formed by phosphorylation of sphingosine, has been implicated in cell growth, suppression of apoptosis, and angiogenesis. In this study, we have examined the contribution of intracellular S1P to tumorigenesis of breast adenocarcinoma MCF-7 cells. Enforced expression of sphingosine kinase type 1 (SPHK1) increased S1P levels and blocked MCF-7 cell death induced by anti-cancer drugs, sphingosine, and TNF-alpha. SPHK1 also conferred a growth advantage, as determined by proliferation and growth in soft agar, which was estrogen dependent. While both ERK and Akt have been implicated in MCF-7 cell growth, SPHK1 stimulated ERK1/2 but had no effect on Akt. Surprisingly, parental growth of MCF-7 cells was only weakly stimulated by S1P or dihydro-S1P, ligands for the S1P receptors which usually mediate growth effects. When injected into mammary fat pads of ovariectomized nude mice implanted with estrogen pellets, MCF-7/SPHK1 cells formed more and larger tumors than vector transfectants with higher microvessel density in their periphery. Collectively, our results suggest that SPHK1 may play an important role in breast cancer progression by regulating tumor cell growth and survival.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Breast Neoplasms; Estrogens; Humans; Lysophospholipids; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neoplasms, Hormone-Dependent; Phosphotransferases (Alcohol Group Acceptor); Receptors, Cell Surface; Receptors, Estrogen; Receptors, G-Protein-Coupled; Receptors, Lysophospholipid; Sphingosine; Tumor Cells, Cultured