sphingosine-1-phosphate and Adenocarcinoma

Although obesity with associated inflammation is now recognized as a risk factor for breast cancer and distant metastases, the functional basis for these connections remain poorly understood. Here, we show that in breast cancer patients and in animal breast cancer models, obesity is a sufficient cause for increased expression of the bioactive sphingolipid mediator sphingosine-1-phosphate (S1P), which mediates cancer pathogenesis. A high-fat diet was sufficient to upregulate expression of sphingosine kinase 1 (SphK1), the enzyme that produces S1P, along with its receptor S1PR1 in syngeneic and spontaneous breast tumors. Targeting the SphK1/S1P/S1PR1 axis with FTY720/fingolimod attenuated key proinflammatory cytokines, macrophage infiltration, and tumor progression induced by obesity. S1P produced in the lung premetastatic niche by tumor-induced SphK1 increased macrophage recruitment into the lung and induced IL6 and signaling pathways important for lung metastatic colonization. Conversely, FTY720 suppressed IL6, macrophage infiltration, and S1P-mediated signaling pathways in the lung induced by a high-fat diet, and it dramatically reduced formation of metastatic foci. In tumor-bearing mice, FTY720 similarly reduced obesity-related inflammation, S1P signaling, and pulmonary metastasis, thereby prolonging survival. Taken together, our results establish a critical role for circulating S1P produced by tumors and the SphK1/S1P/S1PR1 axis in obesity-related inflammation, formation of lung metastatic niches, and breast cancer metastasis, with potential implications for prevention and treatment.

Topics: Adaptor Proteins, Signal Transducing; Adenocarcinoma; Animals; Breast Neoplasms; Cell Line, Tumor; Culture Media, Conditioned; Cytokines; Diet, High-Fat; Disease Models, Animal; Female; Fingolimod Hydrochloride; Humans; Immunosuppressive Agents; Inflammation; Interleukin-6; Lung; Lysophospholipids; Macrophages; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Obesity; Receptors, Lysosphingolipid; Sphingosine; Sphingosine-1-Phosphate Receptors

Gut-associated inflammation plays a crucial role in the progression of colon cancer. Here, we identify a novel pathogen-host interaction that promotes gut inflammation and the development of colon cancer. We find that enteropathogenic bacteria-secreted particles (ET-BSPs) stimulate intestinal epithelium to produce IDENs (intestinal mucosa-derived exosome-like nanoparticles) containing elevated levels of sphingosine-1-phosphate, CCL20 and prostaglandin E2 (PGE2). CCL20 and PGE2 are required for the recruitment and proliferation, respectively, of Th17 cells, and these processes also involve the MyD88-mediated pathway. By influencing the recruitment and proliferation of Th17 cells in the intestine, IDENs promote colon cancer. We demonstrate the biological effect of sphingosine-1-phosphate contained in IDENs on tumour growth in spontaneous and transplanted colon cancer mouse models. These findings provide deeper insights into how host-microbe relationships are mediated by particles secreted from both bacterial and host cells.

Topics: Adenocarcinoma; Animals; Azoxymethane; Bacteroides fragilis; Blotting, Western; Carcinogenesis; Carcinogens; Cell Line, Tumor; Cell Proliferation; Chemokine CCL20; Colitis; Colonic Neoplasms; Dextran Sulfate; Dinoprostone; Disease Models, Animal; Enterobacteriaceae; Exosomes; Immunohistochemistry; In Situ Hybridization, Fluorescence; Inflammation; Intestinal Mucosa; Lysophospholipids; Mice; Myeloid Differentiation Factor 88; Nanoparticles; Neoplasm Transplantation; Reverse Transcriptase Polymerase Chain Reaction; Sphingosine; Th17 Cells

Various experimental studies indicate potential involvement of bone marrow (BM)-derived stem cells (SCs) in malignancy development and progression. In this study, we comprehensively analysed systemic trafficking of various populations of BM-derived SCs (BMSCs), i.e., mesenchymal, haematopoietic, endothelial stem/progenitor cells (MSCs, HSCs, EPCs respectively), and of recently discovered population of very small embryonic/epiblast-like SCs (VSELs) in pancreatic cancer patients. Circulating CD133(+)/Lin(-)/CD45(-)/CD34(+) cells enriched for HSCs, CD105(+)/STRO-1(+)/CD45(-) cells enriched for MSCs, CD34(+)/KDR(+)/CD31(+)/CD45(-) cells enriched for EPCs and small CXCR4(+) CD34(+) CD133(+) subsets of Lin(-) CD45(-) cells that correspond to VSELs were enumerated and sorted from blood samples derived from 29 patients with pancreatic cancer, and 19 healthy controls. In addition, plasma levels of stromal-derived factor-1 (SDF-1), growth/inhibitory factors and sphingosine-1-phosphate (S1P; chemoattractants for SCs), as well as, of complement cascade (CC) molecules (C3a, C5a and C5b-9/membrane attack complex--MAC) were measured. Higher numbers of circulating VSELs and MSCs were detected in pancreatic cancer patients (P < 0.05 and 0.01 respectively). This trafficking of BMSCs was associated with significantly elevated C5a (P < 0.05) and C5b-9/MAC (P < 0.005) levels together with S1P concentrations detected in plasma of cancer patients, and seemed to be executed in a SDF-1 independent manner. In conclusion, we demonstrated that in patients with pancreatic cancer, intensified peripheral trafficking of selected populations of BMSCs occurs. This phenomenon seems to correlate with systemic activation of the CC, hepatocyte growth factor and S1P levels. In contrast to previous studies, we demonstrate herein that systemic SDF-1 levels do not seem to be linked with increased mobilization of stem cells in patients with pancreatic cancer.

Topics: Adenocarcinoma; Aged; Antigens, CD; Biomarkers; Bone Marrow Cells; Case-Control Studies; Cell Movement; Chemokine CXCL12; Complement System Proteins; Female; Gene Expression; Hematopoietic Stem Cells; Humans; Lysophospholipids; Male; Mesenchymal Stem Cells; Middle Aged; Neoplastic Stem Cells; Pancreatic Neoplasms; Sphingosine

The tumor suppressor PTEN is now understood to regulate cellular processes at the cytoplasmic membrane, where it classically regulates PI3K signaling, as well as in the nucleus where multiple roles in controlling cell cycle and genome stability have been elucidated. Mechanisms that dictate nuclear import and, less extensively, nuclear export of PTEN have been described, however the relevance of these processes in disease states, particularly cancer, remain largely unknown. We investigated the impact of acid ceramidase on the nuclear-cytoplasmic trafficking of PTEN. Immunohistochemical analysis of a human prostate tissue microarray revealed that nuclear PTEN was lost in patients whose tumors had elevated acid ceramidase. We found that acid ceramidase promotes a reduction in nuclear PTEN that is dependent upon sphingosine 1-phosphate-mediated activation of Akt. We were further able to show that sphingosine 1-phosphate promotes formation of a complex between Crm1 and PTEN, and that leptomycin B prevents acid ceramidase and sphingosine 1-phosphate mediated loss of nuclear PTEN, suggesting an active exportin-mediated event. To investigate whether the tumor promoting aspects of acid ceramidase in prostate cancer depend upon its ability to export PTEN from the nucleus, we used enforced nuclear expression of PTEN to study docetaxel-induced apoptosis and cell killing, proliferation, and xenoengraftment. Interestingly, while acid ceramidase was able to protect cells expressing wild type PTEN from docetaxel, promote proliferation and xenoengraftment, acid ceramidase had no impact in cells expressing PTEN-NLS. These findings suggest that acid ceramidase, through sphingosine 1-phosphate, promotes nuclear export of PTEN as a means of promoting tumor formation, cell proliferation, and resistance to therapy.

Topics: Acid Ceramidase; Active Transport, Cell Nucleus; Adenocarcinoma; Animals; Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Nucleus; Cytoplasm; Docetaxel; Drug Resistance, Neoplasm; Exportin 1 Protein; Gene Expression Regulation, Neoplastic; Humans; Karyopherins; Lysophospholipids; Male; Mice; Neoplasm Transplantation; Phosphatidylinositol 3-Kinases; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Sphingosine; Taxoids

Macrophages frequently infiltrate tumors and can enhance cancer growth, yet the origins of the macrophage response are not well understood. Here we address molecular mechanisms of macrophage production in a conditional mouse model of lung adenocarcinoma. We report that overproduction of the peptide hormone Angiotensin II (AngII) in tumor-bearing mice amplifies self-renewing hematopoietic stem cells (HSCs) and macrophage progenitors. The process occurred in the spleen but not the bone marrow, and was independent of hemodynamic changes. The effects of AngII required direct hormone ligation on HSCs, depended on S1P(1) signaling, and allowed the extramedullary tissue to supply new tumor-associated macrophages throughout cancer progression. Conversely, blocking AngII production prevented cancer-induced HSC and macrophage progenitor amplification and thus restrained the macrophage response at its source. These findings indicate that AngII acts upstream of a potent macrophage amplification program and that tumors can remotely exploit the hormone's pathway to stimulate cancer-promoting immunity.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Angiotensin II; Animals; Carcinoma, Non-Small-Cell Lung; Cell Communication; Cell Movement; Cell Proliferation; Gene Expression; Hematopoietic Stem Cells; Humans; Lung Neoplasms; Lysophospholipids; Macrophages; Mice; Mice, Transgenic; Signal Transduction; Sphingosine; Spleen; Tumor Burden

Sphingosine-1-phosphate (S1P) regulates a wide array of biological functions. However, the role of S1P signaling in tumorigenesis remains to be elucidated. In this study, we show that S1P receptor subtype 3 (S1P₃) is markedly up-regulated in a subset of lung adenocarcinoma cells compared to normal lung epithelial cells. Specific knockdown of S1P₃ receptors inhibits proliferation and anchorage-independent growth of lung adenocarcinoma cells. Mechanistically, we demonstrate that S1P₃ signaling increases epidermal growth factor receptor (EGFR) expression via the Rho kinase (ROCK) pathway in lung adenocarcinoma cells. Nuclear run-off analysis indicates that S1P/S1P₃ signaling transcriptionally increases EGFR expression. Knockdown of S1P₃ receptors diminishes the S1P-stimulated EGFR expression in lung adenocarcinoma cells. Moreover, S1P treatment greatly enhances EGF-stimulated colony formation, proliferation and invasion of lung adenocarcinoma cells. Together, these results suggest that the enhanced S1P₃-EGFR signaling axis may contribute to the tumorigenesis or progression of lung adenocarcinomas.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Carcinoma, Lewis Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Lysophospholipids; Mice; Neoplasm Invasiveness; Receptors, Lysosphingolipid; rho-Associated Kinases; RNA Interference; RNA, Messenger; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Time Factors; Transcriptional Activation; Transfection; Up-Regulation

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

Phospholipase D (PLD) is involved in the signaling by many extracellular ligands, and its regulation appears to be quite complex. We investigated the signaling pathways initiated by bradykinin (BK) or sphingosine 1-phosphate (S1P) in A549 cells to define molecular mechanisms responsible for their additive effects on PLD activity. BK and S1P each elicited a sustained increase in phosphatidic acid content through a rapid and transient activation of PLD. The two pathways demonstrated rapid homologous downregulation, but heterologous desensitization was not observed. Action of both agonists required protein kinase C (PKC) activation and Ca(2+) influx but was mediated by different heterotrimeric G proteins. In membranes, inhibition of PKCdelta by rottlerin enhanced BK activation of PLD but inhibited that by S1P. Rottlerin inhibited activation of PLD in nuclei by both BK and S1P. By in situ immunofluorescence or cell fractionation followed by immunoblotting, PLD1 was concentrated primarily in nuclei, whereas the membrane fraction contained PLD2 and PLD1. Moreover, PKCdelta specifically phosphorylated recombinant PLD2, but not PLD1. BK and S1P similarly enhanced RhoA translocation to nuclei, whereas BK was less efficacious than S1P on RhoA relocalization to membranes. Effects of both agonists on the nuclear fraction, which contains only PLD1, are compatible with a RhoA- and PKCdelta-dependent process. In membranes, which contain both PLD1 and PLD2, the stimulatory effect of S1P on PLD activity can best be explained by RhoA- and PKCdelta-dependent activation of PLD1; in contrast, the effects of BK on RhoA translocation and enhancement of BK-stimulated PLD activity by PKC inhibition are both consistent with PLD2 serving as its primary target.

Topics: Adenocarcinoma; Bradykinin; Calcium; Enzyme Activation; Glycerophospholipids; Heterotrimeric GTP-Binding Proteins; Humans; Lung Neoplasms; Lysophospholipids; Phosphatidic Acids; Phospholipase D; Protein Kinase C; Protein Kinase C-delta; Protein Transport; rhoA GTP-Binding Protein; Signal Transduction; Sphingosine; Subcellular Fractions; Tritium; 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

In the present paper, the effect of sphingosine 1-phosphate (Sph-1-P) on arachidonic acid mobilization in A549 human lung adenocarcinoma cells was investigated. Sph-1-P provoked a rapid and relevant release of arachidonic acid which was similar to that elicited by bradykinin, well-known pro-inflammatory agonist. The Sph-1-P-induced release of arachidonic acid involved Ca(2+)-independent phospholipase A(2) (iPLA2) activity, as suggested by the dose-dependent inhibition exerted by the rather specific inhibitor bromoenol lactone. The Sph-1-P-induced release of arachidonic acid was pertussis toxin-sensitive, pointing at a receptor-mediated mechanism, which involves heterotrimeric Gi proteins. The action of Sph-1-P was totally dependent on protein kinase C (PKC) catalytic activity and seemed to involve agonist-stimulated phospholipase D (PLD) activity. This study represents the first evidence for Sph-1-P-induced release of arachidonic acid which occurs through a specific signaling pathway involving Gi protein-coupled receptor(s), PKC, PLD and iPLA2 activities.

Topics: Adenocarcinoma; Arachidonic Acid; Enzyme Inhibitors; Humans; Lipid Mobilization; Lung Neoplasms; Lysophospholipids; Phosphatidic Acids; Phospholipase D; Phospholipases A; Protein Kinase C; Sphingosine; Tritium; Tumor Cells, Cultured

Phospholipase D (PLD) has been implicated as a crucial signaling enzyme in secretory pathways. Two 20-kDa guanine nucleotide-binding proteins, Rho and ADP-ribosylation factor (ARF), are involved in the regulation of secretion and can activate PLD in vitro. We investigated in intact (human adenocarcinoma A549 cells) the role of RhoA and ARF in activation of PLD by phorbol 12-myristate 13-acetate, bradykinin, and/or sphingosine 1-phosphate. To express recombinant Clostridium botulinum C3 exoenzyme (using double subgenomic recombinant Sindbis virus C3), an ADP-ribosyltransferase that inactivates Rho, or dominant-negative Rho containing asparagine at position 19 (using double subgenomic recombinant Sindbis virus Rho19N), cells were infected with Sindbis virus, a novel vector that allows rapid, high level expression of heterologous proteins. Expression of C3 toxin or Rho19N increased basal and decreased phorbol 12-myristate 13-acetate-stimulated PLD activity. Bradykinin or sphingosine 1-phosphate increased PLD activity with additive effects that were abolished in cells expressing C3 exoenzyme or Rho19N. In cells expressing C3, modification of Rho appeared to be incomplete, suggesting the existence of pools that differed in their accessibility to the enzyme. Similar results were obtained with cells scrape-loaded in the presence of C3; however, results with virus infection were more reproducible. To assess the role of ARF, cells were incubated with brefeldin A (BFA), a fungal metabolite that disrupts Golgi structure and inhibits enzymes that catalyze ARF activation by accelerating guanine nucleotide exchange. BFA disrupted Golgi structure, but did not affect basal or agonist-stimulated PLD activity, i.e. it did not alter a rate-limiting step in PLD activation. It also had no effect on Rho-stimulated PLD activity, indicating that RhoA action did not involve a BFA-sensitive pathway. A novel PLD activation mechanism, not sensitive to BFA and involving RhoA, was identified in human airway epithelial cells by use of a viral infection technique that preserves cell responsiveness.

Topics: Adenocarcinoma; Adenylyl Cyclases; ADP Ribose Transferases; ADP-Ribosylation Factors; Botulinum Toxins; Bradykinin; Brefeldin A; Golgi Apparatus; GTP-Binding Proteins; GTPase-Activating Proteins; Humans; Lysophospholipids; Phospholipase D; rhoA GTP-Binding Protein; Sphingosine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured