Fig 2: Tumor shows an altered pattern of sphingolipid metabolic enzyme expression as compared with the adjacent normal lung tissue. (a) RT-PCR analysis shows the relative expression of sphingolipid metabolic genes in matched NSCLC patient tumors and adjacent normal lung tissues (n = 10 NSCLC patients). (b) Heatmap represents the Pearson correlation matrix of individual sphingolipid metabolic genes. Rows and columns correspond to the sphingolipid metabolic genes. (c) Gene co-expression study, using COXPRESdb v7 databse, reveals that both B3GNT5 and GAL3ST1 genes coexpressed with the genes that involved in MAPK signaling and sphingolipid signaling pathways. (d) Schematic representation of the sphingolipid metabolic network shows up-regulated (highlight in red) and down-regulated (highlight in green) sphingolipid metabolic genes in NSCLC tumors as compared to the adjacent normal lung tissues. Bar charts represent means ± s.e.m. *p< 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. N.S., no significant difference. (a) Student's t test.

Fig 3: Alteration of sphingolipid metabolic gene expression that involved in the metabolism of bioactive sphingolipids strongly correlates with poor prognosis in NSCLC patients. (a) Metabolic network shows selected sphingolipid metabolic reactions (Kyoto Encyclopedia of Genes and Genomes pathway, KEGG), together with main metabolites (rounded rectangles) and 15 selected sphingolipid metabolic genes (rectangles). Heatmap analysis of the RNA-seq datasets obtained from TCGA, GEO and Hou lung indicates a consistent alteration of 15 sphingolipid metabolic gene expression in NSCLC patient tissues as compared to the normal lung tissues, across all three databases. (GEO: n = 70 normal lung tissues, n = 1251 tumor tissues; TCGA database: n = 59 normal lung tissues, n = 535 tumor tissues; Hou lung database: n=65 normal lung tissues, n = 91 tumor tissues). Table shows the average fold change and statistical analysis of 15 sphingolipid metabolic gene expression between tumor tissues and normal lung tissues obtained from the three databases. (b) Pearson correlation matrix of individual sphingolipid metabolic genes. Rows and columns correspond to the 15 measured enzymes. Each colored square illustrates the gene-gene interaction; color code: red, positive correlation; blue, negative correlation; the color intensity represents the correlation strength. The values range between -1.0 and 1.0. A correlation of -1.0 shows a perfect negative correlation, while a correlation of 1.0 demonstrates a perfect positive correlation. A correlation of 0.0 indicates no relationship between the two genes. (c) Violin plots show the relative mRNA expression level of CERS3, ASAH1, SPHK1, CERK, UGT8, GAL3ST1, B4GALT6 or B3GNT5 in the tumors derived from early stage (stage I) and advanced stage (stage ?/?) respectively (n=223 early stage (stage I) NSCLC patients, n = 179 advanced stage (stage ?/?) NSCLC patients). The datasets are obtained from TCGA database. Values are shown as log2 fold-change relative to normal lung tissues. Violin plots represent means ± s.e.m. (d) Kaplan-Meier curves of overall survival in relation to the CERS3, ASAH1, SPHK1, CERK, UGT8, GAL3ST1, B4GALT6 and B3GNT5 gene expression. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. NS, no significant difference. (a, c) Student's t test. (d) Log-rank test.

Fig 4: Aberrant expression of B3GNT5 or GAL3ST1 regulates cancer cell growth, migration, invasion and sphere formation abilities and in vivo tumor growth. (a) Western blot analysis of stable B3GNT5/GAL3ST1 expressing cells and empty vector transfected cells (ctrl) as indicated. Molecular weight markers (in kDa) are shown on the right. (b, c) CCK8 proliferation assays of stable B3GNT5/GAL3ST1 expressing cells and empty vector transfected cells. Bar charts indicate the relative proliferation of stable B3GNT5/GAL3ST1 expressing cells as compared to empty vector transfected cells. (d, e) Colony formation assays of stable B3GNT5/GAL3ST1 expressing cells and empty vector transfected cells. Representative pictures of colony formation from each group are given. (f, g) Wound healing assay of stable B3GNT5/GAL3ST1 expressing cells and empty vector transfected cells. Bar charts represent the relative wound closure of stable B3GNT5/GAL3ST1 expressing cells as compared to empty vector transfected cells. Representative images of wound healing assays from each group are given. (h, i) Transwell invasion assays. Overexpression of B3GNT5 increased the invasion ability of A549/HCC827 cells, while GALST3 overexpression inhibited PC9/H1299 cell invasion as compared to empty vector control cells. Representative images of crystal violet stained invaded cells from each group are given. (j, k) Sphere formation assays of stable B3GNT5/GAL3ST1 expressing cells and empty vector transfected cells. Bar charts indicate the number of tumor spheres per group (n = 3 experimental repeats). (l, m) Nude mice were subcutaneously injected with either stable B3GNT5 expressing cells (A549-B3GTN5), empty vector transfected A549 cells (A549-Ctrl), stable GAL3ST1 expressing PC9 cells (PC9-GAL3ST1) or empty vector transfected PC9 cells (PC9-ctrl). Tumor growth curves showed that B3GNT5 overexpression increased the tumor growth of A549 cells as compared with empty vector transfected cells, while overexpression of GAL3ST1 repressed the tumor growth of PC9 cells when compared with empty vector transfected cells (ctrl) (n = 5 mice per group). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. NS, no significant difference. (b-k) Student's t test. (l, m) Two-way ANOVA test. Scale bars in (f-k) represent 100 µm.

Fig 5: Sphingolipidomics analysis of B3GNT5/GAL3ST1 genetically perturbed lung cancer cells confirms the circular sphingolipid coregulatory network. (a-d) Manhattan plot analysis of the relative levels of clinically relevant sphingolipid subclasses, including galactosylceramide (GalCer)/glucosylceramide (GlcCer), sulfatide, lactosylceramide (LacCer), lacto/neolacto-series glycosphingolipid in B3GNT5/GAL3ST1 stably overexpressed A549/PC9 cells or B3GNT5/GAL3ST1 stably knock-down A549/PC9 cells. Data are normalized to the levels of these sphingolipid subclasses in empty vector transfected cells or scramble control transfected cells. (e) Heatmap represents the Pearson correlation matrix of individual sphingolipid metabolic genes. Rows and columns correspond to the sphingolipid metabolic genes. Black dotted boxes show clusters of strongly positively correlated sphingolipid metabolic genes. (f) Network visualization of the sphingolipid-sphingolipid correlations in cancer cells. Nodes represent each sphingolipid species. Colors represent sphingolipid subclasses. Edges are correlations of r = 0.7. (g) Network close-up reveals the strongly correlated lacto/neolacto-series glycosphingolipid and sulfatide species distributed at the center of the sphingolipid coregulatory network. (h, j) Nodes of the network are color-coded based on the relative fold change of sphingolipid abundance for B3GNT5/GAL3ST1 stably expressing cells after normalized to empty vector transfected cells. (i, k) Bar charts indicate that overexpression of B3GNT5/GAL3ST1 in A549 or PC9 cells increases the relative abundance levels of lacto/neolacto-series glycosphingolipid and sulfatide species as compared to empty vector transfected cells (ctrl) (n = 3 experimental repeats). Bar charts represent means ± s.e.m. **p<0.01; ***p<0.001; ****p<0.0001. (a-d, i, k) Student's t test.