i(3)so3-galactosylceramide and Kidney-Neoplasms

Sulfatide synthesis in the human renal cancer cell line SMKT-R3 was strongly inhibited in the presence of low µM concentrations of AG-205, a progesterone receptor membrane component 1 (PGRMC1) antagonist. This was also the case in Chinese hamster ovary (CHO) cells stably transfected with UDP-galactose: ceramide galactosyltransferase and cerebroside sulfotransferase, the two enzymes required for sulfatide synthesis. In CHO cells synthesizing galactosylceramide but not sulfatide, galactosylceramide was also strongly reduced, suggesting an effect at the level of galactolipid synthesis. Notably, AG-205 inhibited galactosylceramide synthesis to a similar extent in wild type CHO cells and cells that lack PGRMC1 and/or PGRMC2. In vitro enzyme activity assays showed that AG-205 is an inhibitor of UDP-galactose: ceramide galactosyltransferase, but not cerebroside sulfotransferase. This study shows that PGRMC1 is only one of several targets of AG-205 and should be used with caution, especially in studies using cells synthesizing galactosylceramide and sulfatide.

Topics: Animals; CHO Cells; Cricetulus; Enzyme Inhibitors; Galactosylceramides; Humans; Indoles; Kidney Neoplasms; Membrane Proteins; N-Acylsphingosine Galactosyltransferase; Receptors, Progesterone; Sulfoglycosphingolipids; Sulfotransferases; Uridine Diphosphate Galactose

Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer and the major cause of mortality for individuals with von Hippel-Lindau (VHL) disease. ccRCC is characterized most frequently by inactivation of VHL tumor suppressor protein that mediates degradation of the alpha subunit of the hypoxia-inducible factor (HIF) transcription factor family. HIF has been implicated in disease progression and the aim of this study was to identify novel HIF target genes that may contribute to ccRCC. We show that GAL3ST1, an enzyme that catalyzes the sulfonation of the plasma membrane sulfolipid sulfatide, is among the top 50 upregulated genes in ccRCC tissue relative to matched normal tissue. Increased expression of GAL3ST1 in primary ccRCC correlates with decreased survival. We show that GAL3ST1 is a HIF target gene whose expression is induced upon VHL loss leading to the accumulation of its enzymatic product sulfatide. Notably, platelets bind more efficiently to renal cancer cells with high GAL3ST1-sulfatide expression than to GAL3ST1-sulfatide-negative counterparts, which protects ccRCC cells against natural killer cell-mediated cytotoxicity. These results suggest that GAL3ST1 is a HIF-responsive gene that may contribute to ccRCC development via promoting cancer cell evasion of immune surveillance. IMPLICATIONS: Cancer development is in part dependent on evasion of immune response. We identify a HIF target gene product GAL3ST1 that may play a role in this critical process.

Topics: Apoptosis; Blood Platelets; Carcinoma, Renal Cell; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Immune Evasion; Kidney Neoplasms; Killer Cells, Natural; Microscopy, Fluorescence; Models, Biological; Sulfoglycosphingolipids; Sulfotransferases; Tumor Cells, Cultured; Up-Regulation; Von Hippel-Lindau Tumor Suppressor Protein

Matrix-assisted laser desorption/ionization coupled with Orbitrap mass spectrometry (MALDI-Orbitrap-MS) is used for the clinical study of patients with renal cell carcinoma (RCC), as the most common type of kidney cancer. Significant changes in sulfoglycosphingolipid abundances between tumor and autologous normal kidney tissues are observed. First, sulfoglycosphingolipid species in studied RCC samples are identified using high mass accuracy full scan and tandem mass spectra. Subsequently, optimization, method validation, and statistical evaluation of MALDI-MS data for 158 tissues of 80 patients are discussed. More than 120 sulfoglycosphingolipids containing one to five hexosyl units are identified in human RCC samples based on the systematic study of their fragmentation behavior. Many of them are recorded here for the first time. Multivariate data analysis (MDA) methods, i.e., unsupervised principal component analysis (PCA) and supervised orthogonal partial least square discriminant analysis (OPLS-DA), are used for the visualization of differences between normal and tumor samples to reveal the most up- and downregulated lipids in tumor tissues. Obtained results are closely correlated with MALDI mass spectrometry imaging (MSI) and histologic staining. Important steps of the present MALDI-Orbitrap-MS approach are also discussed, such as the selection of best matrix, correct normalization, validation for semiquantitative study, and problems with possible isobaric interferences on closed masses in full scan mass spectra. Graphical Abstract ᅟ.

Topics: Biomarkers, Tumor; Carcinoma, Renal Cell; Humans; Kidney; Kidney Neoplasms; Least-Squares Analysis; Multivariate Analysis; Principal Component Analysis; Sensitivity and Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sulfoglycosphingolipids

The nature of the regulatory cell types that dominate in any given tumor is not understood at present. Here, we addressed this question for regulatory T cells (Treg) and type II natural killer T (NKT) cells in syngeneic models of colorectal and renal cancer. In mice with both type I and II NKT cells, or in mice with neither type of NKT cell, Treg depletion was sufficient to protect against tumor outgrowth. Surprisingly, in mice lacking only type I NKT cells, Treg blockade was insufficient for protection. Thus, we hypothesized that type II NKT cells may be neutralized by type I NKT cells, leaving Tregs as the primary suppressor, whereas in mice lacking type I NKT cells, unopposed type II NKT cells could suppress tumor immunity even when Tregs were blocked. We confirmed this hypothesis in 3 ways by reconstituting type I NKT cells as well as selectively blocking or activating type II NKT cells with antibody or the agonist sulfatide, respectively. In this manner, we showed that blockade of both type II NKT cells and Tregs is necessary to abrogate suppression of tumor immunity, but a third cell, the type I NKT cell, determines the balance between these regulatory mechanisms. As patients with cancer often have deficient type I NKT cell function, managing this delicate balance among 3 T-cell subsets may be critical for the success of immunotherapy for human cancer.

Topics: Animals; Cell Line, Tumor; Colorectal Neoplasms; Female; Kidney Neoplasms; Mice; Mice, Inbred BALB C; Natural Killer T-Cells; Sulfoglycosphingolipids; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory

Hepatocyte growth factor (HGF) is a heparin-binding pleiotropic factor that acts on a variety of epithelial cells. The interaction of human HGF with glycolipids was studied by overlaying them with 125I-HGF on thin layer chromatograms and by a solid-phase assay using lipids adsorbed on microtiter plates. Among various glycolipids tested, HGF was found to bind to sulfoglycolipids, including galactosylceramide sulfate (SM4), lactosylceramide sulfate (SM3), and gangliotriaosylceramide bis-sulfate. In contrast, HGF failed to bind to gangliosides or neutral glycolipids. HGF binding to SM4 was strongly inhibited by dextran sulfate, heparin, and fucoidan, whereas neither keratan sulfate nor hyaluronic acid had any inhibitory activity. When glycolipids from a renal cancer cell line, SMKT-R3, which overexpresses sulfoglycolipids, were developed on a thin layer chromatogram, SM4 and SM3 were the only glycolipids that bound HGF. We further examined the effect of the incorporation of glycolipids into SMKT-R3 cells on HGF binding to the cells. The incorporation of SM4 into the cells enhanced HGF binding to SMKT-R3 cells, while that of galactosylceramide, a precursor of SM4, had no effect. These observations indicated that SM4 exogenously incorporated into the cell membranes could react with HGF and suggested that endogenous sulfoglycolipids on SMKT-R3 cells might function as reservoirs for HGF.

Topics: Carcinoma, Renal Cell; Cell Line; Cell Membrane; Chromatography, Thin Layer; Glycolipids; Hepatocyte Growth Factor; Humans; Iodine Radioisotopes; Kidney Neoplasms; Kinetics; Membrane Lipids; Sulfoglycosphingolipids; Tumor Cells, Cultured

Lactosylceramide sulfate and galactosylceramide sulfate were found to be increased markedly in human renal cell carcinoma (adenocarcinoma) as compared to uninvolved tissue, while neither of them were found in human nephroblastoma tissues. Activities of two sulfotransferases toward galactosyl ceramide and lactosylceramide as substrates were significantly elevated in the renal cell carcinoma compared to the uninvolved, endorsing enhanced synthesis of the two sulfatides in the renal cell carcinoma. The levels of elevated activities of two sulfotransferases were parallel in most renal cell carcinomas. In nephroblastoma tissues, the activity of sulfotransferase was not detected or only in trace, if any. No consistent change in the activity of arylsulfatase A which disulfate two sulfatides was observed in nephroblastoma and renal cell carcinoma as compared to that in the uninvolved tissue. In the nephroblastoma and the renal cell carcinoma, neolactosylceramide was detected but not in the uninvolved tissue. The present results show that the increased sulfatide (s) in the renal cell carcinoma and the disappearance of the sulfatides in the nephroblastoma are biochemical characteristics of histologically different carcinoma.

Topics: Arylsulfatases; Carcinoma, Renal Cell; Glycolipids; Humans; Kidney Neoplasms; Sulfoglycosphingolipids; Sulfotransferases; Wilms Tumor

Lactosylceramide sulfate and galactosylceramide sulfate were found to increase markedly in human renal cell carcinoma (adenocarcinoma) as compared to uninvolved tissue. Activities of two sulfotransferases toward galactosylceramide and lactosylceramide as substrates were significantly elevated in the carcinoma compared to the uninvolved tissue resulting in enhanced synthesis of the two sulfatides in the carcinoma. The elevation of the two sulfotransferases was parallel in most tumors, suggesting that the same enzyme is responsible for the enhanced synthesis of two sulfatides. No consistent difference in the activity of arylsulfatase A, which desulfates the two sulfatides, was observed between the carcinoma and uninvolved tissue. Both the present and previous results show that the increased synthesis of the sulfatide(s) due to elevated sulfotransferase activity could be a biochemical characteristic common to adenocarcinomas derived from different tissues.

Topics: Adenocarcinoma; Adult; Aged; Antigens, CD; Carcinoma, Renal Cell; Cerebroside-Sulfatase; Female; Galactosylceramides; Glycosphingolipids; Humans; Kidney Neoplasms; Lactosylceramides; Male; Middle Aged; Sulfoglycosphingolipids; Sulfurtransferases