sphingosine-1-phosphate and Kidney-Neoplasms

Sphingosine 1-phosphate (S1P) is a lipid mediator with numerous biological functions. The term 'S1P' mainly refers to the sphingolipid molecule with a long-chain sphingoid base of 18 carbon atoms, d18:1 S1P. The enzyme serine palmitoyltransferase catalyses the first step of the sphingolipid de novo synthesis using palmitoyl-CoA as the main substrate. After further reaction steps, d18:1 S1P is generated. However, also stearyl-CoA or myristoyl-CoA can be utilised by the serine palmitoyltransferase, which at the end of the S1P synthesis pathway, results in the production of d20:1 S1P and d16:1 S1P respectively. We measured these S1P homologues in mice and renal tissue of patients suffering from renal cell carcinoma (RCC). Our experiments highlight the relevance of d16:1 S1P for the induction of connective tissue growth factor (CTGF) in the human renal clear cell carcinoma cell line A498 and human RCC tissue. We show that d16:1 S1P versus d18:1 and d20:1 S1P leads to the highest CTGF induction in A498 cells via S1P2 signalling and that both d16:1 S1P and CTGF levels are elevated in RCC compared to adjacent healthy tissue. Our data indicate that d16:1 S1P modulates conventional S1P signalling by acting as a more potent agonist at the S1P2 receptor than d18:1 S1P. We suggest that elevated plasma levels of d16:1 S1P might play a pro-carcinogenic role in the development of RCC via CTGF induction.

Topics: Animals; Carbon; Carcinoma, Renal Cell; Connective Tissue Growth Factor; Humans; Kidney Neoplasms; Lysophospholipids; Mice; Receptors, Lysosphingolipid; Serine C-Palmitoyltransferase; Sphingolipids; Sphingosine; Sphingosine-1-Phosphate Receptors

Topics: Angiomyolipoma; Animals; Apoptosis; Ataxin-1; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Female; Humans; Imidazoles; Kidney Neoplasms; Lysophospholipids; Mice; Mice, Inbred NOD; Mice, Knockout; Pyrimidines; Signal Transduction; Sphingosine; Tuberous Sclerosis; Tuberous Sclerosis Complex 2 Protein; Tumor Cells, Cultured

Inhibition of VEGFR signaling is an effective treatment for renal cell carcinoma, but resistance continues to be a major problem. Recently, the sphingosine phosphate (S1P) signaling pathway has been implicated in tumor growth, angiogenesis, and resistance to antiangiogenic therapy. S1P is a bioactive lipid that serves an essential role in developmental and pathologic angiogenesis via activation of the S1P receptor 1 (S1P1). S1P1 signaling counteracts VEGF signaling and is required for vascular stabilization. We used

Topics: Angiogenesis Inhibitors; Animals; Antibodies, Monoclonal; Carcinoma, Renal Cell; Cell Line, Tumor; Drug Therapy, Combination; Endothelial Cells; Female; Humans; Kidney Neoplasms; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Mice, Nude; Neovascularization, Pathologic; Sphingosine; Sphingosine-1-Phosphate Receptors; Sunitinib; Treatment Outcome; Tumor Burden; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2; Xenograft Model Antitumor Assays

VEGFR2 tyrosine kinase inhibition (TKI) is a valuable treatment approach for patients with metastatic renal cell carcinoma (RCC). However, resistance to treatment is inevitable. Identification of novel targets could lead to better treatment for patients with TKI-naïve or -resistant RCC.. In this study, we performed transcriptome analysis of VEGFR TKI-resistant tumors in a murine model and discovered that the SPHK-S1P pathway is upregulated at the time of resistance. We tested sphingosine-1-phosphate (S1P) pathway inhibition using an anti-S1P mAb (sphingomab), in two mouse xenograft models of RCC, and assessed tumor SPHK expression and S1P plasma levels in patients with metastatic RCC.. Resistant tumors expressed several hypoxia-regulated genes. The SPHK1 pathway was among the most highly upregulated pathways that accompanied resistance to VEGFR TKI therapy. SPHK1 was expressed in human RCC, and the product of SPHK1 activity, S1P, was elevated in patients with metastatic RCC, suggesting that human RCC behavior could, in part, be due to overproduction of S1P. Sphingomab neutralization of extracellular S1P slowed tumor growth in both mouse models. Mice bearing tumors that had developed resistance to sunitinib treatment also exhibited tumor growth suppression with sphingomab. Sphingomab treatment led to a reduction in tumor blood flow as measured by MRI.. Our findings suggest that S1P inhibition may be a novel therapeutic strategy in patients with treatment-naïve RCC and also in the setting of resistance to VEGFR TKI therapy.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Cell Line, Tumor; Cluster Analysis; Disease Models, Animal; Drug Resistance, Neoplasm; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; Kidney Neoplasms; Lysophospholipids; Mice; Neoplasm Metastasis; Neovascularization, Pathologic; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Receptors, Vascular Endothelial Growth Factor; Sphingosine; Transcriptome; Tumor Burden; Up-Regulation; Xenograft Model Antitumor Assays

Sphingosine kinase 1 (SK1), the enzyme responsible for sphingosine 1-phosphate (S1P) production, is overexpressed in many human solid tumors. However, its role in clear cell renal cell carcinoma (ccRCC) has not been described previously. ccRCC cases are usually associated with mutations in von Hippel-Lindau (VHL) and subsequent normoxic stabilization of hypoxia-inducible factor (HIF). We previously showed that HIF-2α up-regulates SK1 expression during hypoxia in glioma cells. Therefore, we hypothesized that the stabilized HIF in ccRCC cells will be associated with increased SK1 expression. Here, we demonstrate that SK1 is overexpressed in 786-0 renal carcinoma cells lacking functional VHL, with concomitant high S1P levels that appear to be HIF-2α mediated. Moreover, examining the TCGA RNA seq database shows that SK1 expression was ∼2.7-fold higher in solid tumor tissue from ccRCC patients, and this was associated with less survival. Knockdown of SK1 in 786-0 ccRCC cells had no effect on cell proliferation. On the other hand, this knockdown resulted in an ∼3.5-fold decrease in invasion, less phosphorylation of focal adhesion kinase (FAK), and an ∼2-fold decrease in angiogenesis. Moreover, S1P treatment of SK1 knockdown cells resulted in phosphorylation of FAK and invasion, and this was mediated by S1P receptor 2. These results suggest that higher SK1 and S1P levels in VHL-defective ccRCC could induce invasion in an autocrine manner and angiogenesis in a paracrine manner. Accordingly, targeting SK1 could reduce both the invasion and angiogenesis of ccRCC and therefore improve the survival rate of patients.

Topics: Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Renal Cell; Cell Line, Tumor; Down-Regulation; Focal Adhesion Kinase 1; Gene Knockdown Techniques; Humans; Kidney Neoplasms; Lysophospholipids; Mutation; Neoplasm Invasiveness; Neovascularization, Pathologic; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Sphingosine; Sphingosine-1-Phosphate Receptors; Up-Regulation; Von Hippel-Lindau Tumor Suppressor Protein

MALDI MS imaging (MALDI-MSI) offers a capability to not only evaluate the distribution, localization and metabolism of drugs within tissues but also allow correlative tissue measurement of the effect of the drug on biomolecules in the targeted pathway. Particularly for MALDI-MSI, lipid molecules are readily detectable within tissues. Case study examples are provided for two different drugs targeting the sphingosine-1-phosphate/ceramide nexus in tumor xenograft tissues. A workflow combining high-resolution MALDI-MSI with on-tissue confirmation of targeted compounds using a structural library and on-tissue enzymatic digestion strategy is described. Representative images of drug metabolite distribution that correlate to an increase or decrease in sphingosine-1-phosphate or ceramide species are provided.

Topics: Adamantane; Animals; Biomarkers, Tumor; Ceramides; Disease Models, Animal; Humans; Kidney Neoplasms; Lysophospholipids; Mice; Pancreatic Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Pyridines; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sphingosine; Transplantation, Heterologous

Sphingosine-1-phosphate (S1P) is an important regulator of cellular functions via interaction with its receptors S1P(1-5). To date, nothing is known about the S1P receptor expression and the effects of S1P signaling in Wilms tumor. In this study, we found ubiquitous expression of S1P receptors in Wilms tumor specimens and cell lines. We demonstrated that S1P(1) acted as a promigratory modulator by employing S1P(1) antagonist VPC44116, S1P(1) siRNA and adenoviral transduction in Wilms tumor cells. Further, we clarified that S1P(1)-mediated migration occurred via Gi coupling and activation of PI3K and Rac1. In addition, S1P stimulated WiT49 cell invasion through S1P(1)/Gi signaling pathway. We consider that targeting S1P(1) may be a point of therapeutic intervention in Wilms tumor.

Topics: Cell Line, Tumor; Cell Movement; GTP-Binding Protein alpha Subunits, Gi-Go; Humans; Kidney Neoplasms; Lysophospholipids; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; rac1 GTP-Binding Protein; Receptors, Lysosphingolipid; RNA, Small Interfering; Signal Transduction; Sphingosine; Wilms Tumor

Connective tissue growth factor (CTGF), a member of the CCN family of secreted matricellular proteins, regulates fibrosis, angiogenesis, cell proliferation, apoptosis, tumor growth, and metastasis. However, the role of CTGF and its regulation mechanism in Wilms' tumor remains largely unknown. We found that the bioactive lipid sphingosine-1-phosphate (S1P) induced CTGF expression in a concentration- and time-dependent manner in a Wilms' tumor cell line (WiT49), whereas FTY720-phosphate, an S1P analogue that binds all S1P receptors except S1P2, did not. Further, the specific S1P2 antagonist JTE-013 completely inhibited S1P-induced CTGF expression, whereas the S1P1 antagonist VPC44116 did not, indicating that this effect was mediated by S1P2. This was confirmed by adenoviral transduction of S1P2 in WiT49 cells, which showed that overexpression of S1P2 increased the expression of CTGF. Induction of CTGF by S1P was sensitive to ROCK inhibitor Y-27632 and c-Jun NH2-terminal kinase inhibitor SP600125, suggesting the requirement of RhoA/ROCK and c-Jun NH2-terminal kinase pathways for S1P-induced CTGF expression. Interestingly, the expression levels of CTGF were decreased in 8 of 10 Wilms' tumor tissues compared with matched normal tissues by quantitative real-time PCR and Western blot analysis. In vitro, human recombinant CTGF significantly inhibited the proliferation of WiT49 cells. In addition, overexpression of CTGF resulted in significant inhibition of WiT49 cell growth. Taken together, these data suggest that CTGF protein induced by S1P2 might act as a growth inhibitor in Wilms' tumor.

Topics: Cell Line, Tumor; Cell Proliferation; Connective Tissue Growth Factor; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; JNK Mitogen-Activated Protein Kinases; Kidney Neoplasms; Lysophospholipids; p38 Mitogen-Activated Protein Kinases; Receptors, Lysosphingolipid; rho-Associated Kinases; rhoA GTP-Binding Protein; Sphingosine; Suppression, Genetic; Wilms Tumor