Potential Role of Sphingolipidoses-Associated Lysosphingolipids in Cancer

Abstract

Sphingolipids play a key structural role in cellular membranes and/or act as signaling molecules. Inherited defects of their catabolism lead to lysosomal storage diseases called sphingolipidoses. Although progress has been made toward a better understanding of their pathophysiology, several issues still remain unsolved. In particular, whether lysosphingolipids, the deacylated form of sphingolipids, both of which accumulate in these diseases, are simple biomarkers or play an instrumental role is unclear. In the meanwhile, evidence has been provided for a high risk of developing malignancies in patients affected with Gaucher disease, the most common sphingolipidosis. This article aims at analyzing the potential involvement of lysosphingolipids in cancer. Knowledge about lysosphingolipids in the context of lysosomal storage diseases is summarized. Available data on the nature and prevalence of cancers in patients affected with sphingolipidoses are also reviewed. Then, studies investigating the biological effects of lysosphingolipids toward pro or antitumor pathways are discussed. Finally, original findings exploring the role of glucosylsphingosine in the development of melanoma are presented. While this lysosphingolipid may behave like a protumorigenic agent, further investigations in appropriate models are needed to elucidate the role of these peculiar lipids, not only in sphingolipidoses but also in malignant diseases in general.

Keywords: Gaucher disease; cancer; glucosylsphingosine; lysoGb3; lysosulfatide; melanoma; psychosine; sphingolipid; sphingosylphosphocholine.

Figure 1

Sphingolipid catabolism and associated diseases. Green names correspond to the enzyme names, blue names to their activators, the red boxed texts contain the disease name, and the red and italic names indicate the corresponding lysoSL. Abbreviations: α-Gal: alpha-galactosidase; ACDase, acid ceramidase; Aryl A, arylsulfatase A; β-Gal, beta-galactosidase; Cer, ceramide; GalCase, galactosylceramidase; GalSph, galactosylsphingosine; GCase, glucosylceramidase; GlcCer, glucosylceramide; GlcSph, glucosylsphingosine; Hex, hexosaminidase; LacCer, lactosylceramide; LysoSulf, lysosulfatide; Sap, saposin; SK, sphingosine kinase; SM, sphingomyelin; SMase, sphingomyelinase; Sph, sphingosine; Sulf, sulfatide; S1P, sphingosine 1-phosphate.

Figure 2

Melanoma tumor growth is increased in a Gba1^D409V/D409V Gaucher mouse model. Murine B16F10 melanoma cells (3 × 10⁵) were injected subcutaneously into Gba1^D409V/D409V (D409V/D409V) female mice and heterozygous (D409V/wt) littermates having the same mixed genetic background (19–22 weeks of age). (A) Tumor volumes were measured every 3 days. (B) Tumor weights were measured 25 days after tumor inoculation. Data are expressed as means +/− SEM of at least two independent experiments (n = 7–21 mice). (C) GCase enzyme activity was determined in lysates of liver and spleen isolated from D409V/D409V or heterozygous D409V/wt mice. Assays were performed in duplicate on samples of three to six animals. GlcCer (D) and GlcSph (E) were extracted by the Bligh and Dyer method [146] from the liver and spleen and analyzed by LC-MS (liquid chromatography coupled by mass spectrometry); assays were performed in duplicate on samples taken at day 20 of three to six animals (18–22 weeks of age). Statistical significance was determined by a Mann–Whitney test. *, p < 0.05; **, p < 0.01; ****, p < 0.0001.

Figure 3

GlcSph decreases the growth of cultured human melanoma cells in a dose-dependent manner. (A,B) A375 and WM35 human melanoma cells were seeded into a 96-well plate (10,000 cells/well) and, 24 h later, treated either with different concentrations of GlcSph (1, 2.5, or 5 µM) or with ethanol (veh). Cell growth was assessed by measuring the cellular confluence using an IncuCyte S3 time-lapse microscopy system. For each sample, the cellular confluence was measured in duplicate at different incubation times for 4 days. (C) GCase enzyme activity in A375 cells treated with CBE (30 µM) or DMSO (veh) for 48 h. (D) A375 cells were seeded into a 96-well plate and, after 24 h, treated either with CBE (30 µM) or DMSO (veh). Cell growth was evaluated as described in (A,B). GlcSph (E) and Sph (F) were extracted as described in [13], and concentrations were determined by liquid chromatography-tandem mass spectrometry in CBE- or GlcSph-treated cells compared to controls. For all experiments, data are expressed as means +/− SEM of three independent experiments. Statistical significance was determined by a two-way mixed ANOVA with Geisser–Greenhouse correction (A,B,D) or a Mann–Whitney test (C,E,F). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant compared to vehicle-treated cells.

Figure 4

GlcSph induces necrosis and produces multinucleated cells in A375 human melanoma cells. (A) A375 cells were treated for 24 h with either different concentrations of GlcSph (1, 2.5, and 5 µM) or with ethanol (veh), stained with Annexin V-FITC and 7-AAD, and analyzed by flow cytometry; 20,000 events were recorded and sorted as follows: live cells (Annexin V-negative/7-AAD-negative), dead cells (Annexin V-positive/7-AAD-positive), early apoptosis (Annexin V-positive/7-AAD-negative), and necrosis (Annexin V-negative/7-AAD-positive). Histograms represent the cell frequency of GlcSph-treated cells compared to vehicle-treated cells. (B–E) A375 cells were treated for 48 h with ethanol (B,D) or GlcSph 5 µM (C and E), fixed and stained with phalloidin (for F-actin, green), and nuclei were counterstained with DAPI (for DNA, blue). Then, they were imaged by confocal microscopy (Zeiss) at 63× (B,C), with an additional upper zoom (×2) of the selected regions (white square) (D,E). Scale bar = 20 µm. (F–H) A375 cells were incubated either with different concentrations of GlcSph (1, 2.5, or 5 µM) or with ethanol (veh) for 24, 48, and 72 h. Then, cells were stained with propidium iodide (PI) and analyzed by flow cytometry. Figure (F) is a representative plot of cell cycle analysis of GlcSph- and vehicle-treated A375 cells for 48 h. To identify the peaks corresponding to abnormal ploidies, A375 cells were treated with blebbistatin (10 µM), a myosin II inhibitor (see arrows). The proportion of cells with 8N (G) and 16N (H) in GlcSph-treated cells compared to controls was quantified. For experiments in A, G, and H, data are expressed as means +/− SEM of three independent experiments. Statistical significance was determined by a two-way mixed ANOVA. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001 compared to vehicle-treated cells.