Fig 2: GBA copy number amplifications and increased expression are highly prevalent in human cancers, and are associated with poor outcomes. (a) TCGA data were queried for genomic alterations in GBA (ENSG00000177628) and plotted as the percentage of cases altered in the database by CNV gain alterations (red segments), CNV loss alterations (green segments), and simple somatic mutations (SSM, blue segments). (b) GBA staining profiles of cancers in the Human Protein Atlas. Plot shows the number of cases with high, medium, low, or nondetectable staining of GBA. (c) GBA expression was examined using the TNMplot database. The Mann–Whitney U test determined significant differences between normal and tumor tissue, marked with an asterisk (*). (d) Pan-cancer TCGA data were analyzed for CNV gain alterations in genes of sphingolipid, lacto, neolacto, ganglio, isoglobo, and globo glycosphingolipids, fatty acid synthesis, elongation, and degradation, citrate and pentose phosphate cycles, pyruvate, glycolysis and gluconeogenesis, fructose, mannose, galactose, glycine, serine, threonine, and glycerolipid metabolic pathways. (e) Top 20 genes with CNV gain alterations in (d). (f) TCGA pan-cancer data were examined for genomic alterations in core sphingolipid metabolism genes (Kegg; hsa00600) and plotted as the percentage of subjects with the indicated genetic lesions within the cohort. (g–j) Kaplan–Meier plots generated with the Human Protein Atlas online tool for GBA and the indicated cancers and outcome measures.

Fig 3: Deletion of GBA resulted in the accumulation of GlcCer and LacCer in HeLa and H1703 cells. (a,d) Immunoblots and densitometry analysis with the indicated antibodies of HeLa (a) and H1703 (d) cells in which GBA was deleted using CRISPR/Cas9; n = 3 each line. (b,c,e,f) Mass spectrometry analysis of the indicated lipids in HeLa/HeLa GBAKO (b,c) and H1703/H1703GBAKO (e,f) cells. Lipids were normalized per mg of protein input; n = 3 each line. Plots are bar graphs with the mean ± SEM; t-test analysis (Prism): ns, not significant; * p = 0.05; **** p = 0.0001. Blots were quantified with ImageJ.

Fig 4: HeLa GBAKO and H1703 GBAKO cells have significant reduced expression of EMT markers. Immunoblots (a,d,e) and corresponding densitometry (b,f) analysis of the indicated proteins in HeLa (a,d,e), HeLa cells overexpressing GBA (b,d), and HeLa GBAKO cells (a,e). All cell lines, n = 3. (c) Confocal images of HeLa and HeLa GBAKO cells probed with the indicated antibodies. Nuclei were labeled with DAPI. (g–i) Immunoblots (g,h) and corresponding densitometry analysis (i) of the indicated proteins in H1703 and H1703 GBAKO cells; n = 3 both lines. Plots are bar graphs with the mean ± SEM. In (b), statistical tests were performed with ANOVA with multiple comparisons (Prism); in (f,i), a t-test (Prism) was performed: ns, not significant; * p = 0.05; ** p = 0.01; *** p = 0.005; **** p = 0.0001. Blots were quantified with ImageJ.

Fig 5: GBA depletion reduced the migratory and invasive capacity of HeLa and H1703 cells, but CßE did not affect their invasive capacity. (a,b) Cell migration in HeLa (a) and H1703 (b) was assayed after a linear segment of confluent cells was removed by scratching. Cells were grown at 37 °C, 5% CO2, and imaged at the indicated times; N = 3 for each line. Wound closure was plotted as the wound closure relative to that measured at t = 0 h. (c,d) The growth of HeLa/HeLa GBAKO (c) and H1703/H1703 GBAKO (d) cells was monitored in 96-well plates using WST-8 assays at the indicated times; N = 5 each line. In (a–d), graphs are scatter plots with the mean ± SEM. ANOVA (Prism): ns, not significant; *** p = 0.005. (e,f) Representative images and corresponding quantification of the indicated cell lines that migrated through a Matrigel matrix and were stained with crystal violet. In (f), cells were treated with either DMSO (0.01% w/v) or 35 µM CßE for 10 days with biweekly medium changes. For all panels, cells were pre-starved for 2 h prior to seeding in the upper chamber of a transwell insert containing Matrigel. Cells were allowed to grow for 48 h at 37 °C and 5% CO2. The lower chamber contained medium supplemented with 10% FBS. Plots are bar graphs with the mean ± SEM; n = 3 for each line. t-Test analysis (Prism): *** p = 0.005; **** p = 0.0001.