sphingosine-kinase and Carcinoma--Renal-Cell

To detect the expression of sphingosine kinase 1 (SPHK1) in clear cell renal cell carcinoma (ccRCC) and explore its biological role in the occurrence and development of ccRCC through regulation of fatty acid metabolism.. Using the Cancer Genome Atlas database, SPHK1 expression and its clinical significance were detected in clear cell renal cell carcinoma. Immunohistochemistry was performed to detect SPHK1 expression in RCC samples in our hospital. The connection between the SPHK1 levels and clinicopathological features of patients was assessed. Nile Red was used to detect fatty acids in cells. Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine assays were performed to determine the effect of SPHK1 on renal cell viability and proliferation, respectively. Additionally, the effects of SPHK1 on the proliferation and metastasis of ccRCC were studied using wound healing and Transwell assays. Fatty acids were added exogenously in recovery experiments and western blotting was performed to determine the effect of SPHK1 on fatty acid metabolism in ccRCC. Finally, the effects of SPHK1 on tumor growth were investigated in a xenograft model.. Bioinformatics analysis revealed that SPHK1 expression was upregulated in kidney RCC. OverSPHK1 expression was associated with poor prognosis for ccRCC patients. High SPHK1 expression was detected in human ccRCC. SPHK1 expression was related to clinicopathological features, such as tumor size and Furman grade. Additionally, cell proliferation, migration, and invasion were inhibited in ccRCC cells with low SPHK1 expression. In rescue experiments, proliferation, migration, and invasion were restored. In vivo, reduced SPHK1 levels correlated with lower expression of fatty acid synthase, stearoyl-CoA desaturase 1, and acetyl CoA carboxylase, and slowed tumor growth.. SPHK1 is abnormally overexpressed in human ccRCC. Patients with ccRCC may benefit from treatments that target SPHK1, which may also serve as a prognostic indicator.

Topics: Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Kidney; Kidney Neoplasms; Lipid Metabolism; Prognosis

von Hippel-Lindau (VHL) is a critical tumor suppressor in clear cell renal cell carcinomas (ccRCCs). It is important to identify additional therapeutic targets in ccRCC downstream of VHL loss besides hypoxia-inducible factor 2α (HIF2α). By performing a genome-wide screen, we identified Scm-like with four malignant brain tumor domains 1 (SFMBT1) as a candidate pVHL target. SFMBT1 was considered to be a transcriptional repressor but its role in cancer remains unclear. ccRCC patients with VHL loss-of-function mutations displayed elevated SFMBT1 protein levels. SFMBT1 hydroxylation on Proline residue 651 by EglN1 mediated its ubiquitination and degradation governed by pVHL. Depletion of SFMBT1 abolished ccRCC cell proliferation in vitro and inhibited orthotopic tumor growth in vivo. Integrated analyses of ChIP-seq, RNA-seq, and patient prognosis identified sphingosine kinase 1 (SPHK1) as a key SFMBT1 target gene contributing to its oncogenic phenotype. Therefore, the pVHL-SFMBT1-SPHK1 axis serves as a potential therapeutic avenue for ccRCC.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Carcinoma, Renal Cell; Cell Cycle; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Genome-Wide Association Study; Humans; Kidney Neoplasms; Mice; Mice, Inbred NOD; Mice, SCID; Phosphotransferases (Alcohol Group Acceptor); Prognosis; Prolyl Hydroxylases; Repressor Proteins; Tumor Cells, Cultured; Ubiquitination; Von Hippel-Lindau Tumor Suppressor Protein; Xenograft Model Antitumor Assays

The PI3K-AKT-mTOR cascade is required for renal cell carcinoma (RCC) progression. SC66 is novel AKT inhibitor. We found that SC66 inhibited viability, proliferation, migration and invasion of RCC cell lines (786-O and A498) and patient-derived primary RCC cells. Although SC66blocked AKT-mTORC1/2 activation in RCC cells, it remained cytotoxic in AKT-inhibited/-silenced RCC cells. In RCC cells, SC66 cytotoxicity appears to occur via reactive oxygen species (ROS) production, sphingosine kinase 1inhibition, ceramide accumulation and JNK activation, independent of AKT inhibition. The ROS scavenger N-acetylcysteine, the JNK inhibitor (JNKi) and the anti-ceramide sphingolipid sphingosine-1-phosphate all attenuated SC66-induced cytotoxicity in 786-O cells. In vivo, oral administration of SC66 potently inhibited subcutaneous 786-O xenograft growth in SCID mice. AKT-mTOR inhibition, SphK1 inhibition, ceramide accumulation and JNK activation were detected in SC66-treated 786-O xenograft tumors, indicating that SC66 inhibits RCC cell progression through AKT-dependent and AKT-independent mechanisms.

Topics: Animals; Antineoplastic Agents; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclohexanones; Female; Humans; JNK Mitogen-Activated Protein Kinases; Kidney Neoplasms; Mice, SCID; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinase; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyridines; Reactive Oxygen Species; Signal Transduction; TOR Serine-Threonine Kinases; Tumor Burden; Xenograft Model Antitumor Assays

Clear cell renal cell carcinoma (ccRCC) is the most common type of human malignancies of the urological system. Soyasapogenol B (Soy B), an ingredient of soybean, has been found to exert anti-proliferative activities in vitro in human breast cancer cells. Our current study aimed to evaluate the effectiveness of Soy B against ccRCC. The effect of Soy B on cell viability was assessed by Cell Counting Kit-8 (CCK-8) assay. The effect of Soy B on cell proliferation was determined by colony formation assay. Apoptotic percentage was determined by flow cytometry following annexin V-FITC/propidium iodide (PI) double staining. JC-1 staining was performed to examine the change in mitochondrial membrane potential. Western blotting was used to determine the level of relevant proteins. Isobaric tags for relative and absolute quantification (iTRAQ) was then performed to identify the potential targets of Soy B. Quantitative real-time PCR (qRT-PCR) was performed to determine the mRNA level of sphingosine kinase 1 (SphK1). The SphK1 expression in ccRCC tissue from patients was examined by immunohistochemistry (IHC) assay. To validate the role of SphK1 involved in the pro-apoptotic activities of Soy B, overexpressed SphK1 vectors and shRNA targeting of SphK1 were utilized to transfected ccRCC cells. Moreover, a ccRCC xenograft murine model was used to analyze the therapeutic efficacy of Soy B in vivo. Soy B incubation led to a decrease in the number of viable cells in ccRCC cell lines and primary ccRCC cells. Soy B also suppressed the proliferation of two model ccRCC cell lines. Soy B promoted apoptotic cell death in a caspase-dependent manner. Moreover, our results showed that both extrinsic and intrinsic apoptotic signaling pathways were involved in Soy B-induced apoptosis. ITRAQ analysis identified SphK1 as most profoundly altered after the treatment of Soy B in ACHN cells. The mediatory role of SphK1 was validated when the pro-apoptotic activity of Soy B was significantly blocked by SphK1 overexpression, while SphK1 knockdown sensitized the ccRCC cells to Soy B. Moreover, in vivo studies also showed that Soy B could exhibit anti-cancer activities against ccRCC. Soy B triggers apoptotic cell death in vitro and in vivo in ccRCC by down-regulating SphK1. Our results highlight the possibility of using Soy B as a chemotherapeutic agent in the prevention and treatment of ccRCC.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Renal Cell; Cell Proliferation; Female; Humans; Kidney; Kidney Neoplasms; Male; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Oleanolic Acid; Phosphotransferases (Alcohol Group Acceptor); Saponins; Tumor Cells, Cultured

Clear cell renal cell carcinoma (ccRCC) is characterized by intratumoral hypoxia and chemoresistance. The hypoxia-inducible factors HIF1α and HIF2α play a crucial role in ccRCC initiation and progression. We previously identified the sphingosine kinase 1/sphingosine 1-phosphate (SphK1/S1P) pathway as a new modulator of HIF1α and HIF2α under hypoxia in various cancer cell models. Here, we report that FTY720, an inhibitor of the S1P signaling pathway, inhibits both HIF1α and HIF2α accumulation in several human cancer cell lines. In a ccRCC heterotopic xenograft model, we show that FTY720 transiently decreases HIF1α and HIF2α intratumoral level and modifies tumor vessel architecture within 5 days of treatment, suggesting a vascular normalization. In mice bearing subcutaneous ccRCC tumor, FTY720 and a gemcitabine-based chemotherapy alone display a limited effect, whereas, in combination, there is a significant effect on tumor size without toxicity. Noteworthy, administration of FTY720 for 5 days before chemotherapy is not associated with a more effective tumor control, suggesting a mode of action mainly independent of the vascular remodeling. In conclusion, these findings demonstrate that FTY720 could successfully sensitize ccRCC to chemotherapy and establish this molecule as a potent therapeutic agent for ccRCC treatment, independently of drug scheduling. Mol Cancer Ther; 15(10); 2465-74. ©2016 AACR.

Topics: Animals; Antineoplastic Agents; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Drug Resistance, Neoplasm; Female; Fingolimod Hydrochloride; Gene Expression; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Lysophospholipids; Mice; Neovascularization, Pathologic; Oxygen Consumption; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Vascular Endothelial Growth Factor A; Vascular Remodeling; 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

Multi-target tyrosine kinase inhibitor Sunitinib has been widely used in cancer treatment, including metastatic renal cell carcinoma. However, most patients who initially benefit from Sunitinib develop resistance with extended usage of Sunitinib, which is referred to as "acquired resistance". The molecular mechanisms contributing to this acquired resistance remain poorly understood. In this present study, we established Sunitinib-resistant cell lines from human renal cell lines (786-O, A498, ACHN and CAKI1) by continuous treatment with Sunitinib to explore the molecular mechanism leading to Sunitinib resistance. We found that PDGFR-β expression in cell seems to be a protective factor against Sunitinib resistance formation. In addition, we found that both SK1 and ERK were activated in Sunitinib-resistance cell lines and SK1 and ERK inhibitors could resensitize Sunitinib-resistant cell lines. In conclusion, our observations suggest that SK1 and ERK activation is a feature of resistant cell lines, which serves as an alternative pathway evading anti-tumor activity of Sunitinib.

Topics: Antineoplastic Agents; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Humans; Indoles; Kidney Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Pyrroles; Receptor, Platelet-Derived Growth Factor beta; Sunitinib