Article Figures & Data
Figures
- Figure 1.
GAL3ST1 expression is upregulated in ccRCC. A, Patients were separated into two groups based on FPKM value for GAL3ST1. Kaplan–Meier estimator measured survival outcomes in patients expressing high or low GAL3ST1 mRNA and outcomes of the two groups were compared with log-rank tests [log-rank P = 1.79e-4 (prognostic, unfavorable)]. B, Affymetrix microarray expression analysis of primary ccRCC (CC), chromophobe (CH), oncocytoma (ON), type I PRCC (P1), type II PRCC (P2), and matched-normal (NO) tissue samples. Top and bottom box boundaries represent the 75th and 25th percentiles, respectively, while the line within the box represents the median of the dataset. Statistical differences relative to normal expression were analyzed using an unpaired t test; the results of these tests are expressed as P values above the designated tumor type. C, Equal amounts of primary ccRCC tumor lysates (T) and matched-normal tissue lysates (N) from six different subjects were immunoblotted (IB) with the indicated antibodies. * indicates nonspecific protein band, while the arrow and bracket indicate GAL3ST1 protein bands.
- Figure 2.
GAL3ST1 is a HIF target gene. A, GAL3ST1 and GLUT1 mRNA from 786 or RCC4-MOCK and -VHL cells was quantified using qRT-PCR. B, 786 cells stably expressing empty MOCK plasmid, WT HA-VHL, HA-VHL (C162F), and HA-VHL (L188V) were lysed and immunoblotted with the indicated antibodies. C, 786 and RCC4-MOCK and -VHL cells were grown in normoxia (21% O2) or hypoxia (1% O2) for 48 hours. GAL3ST1 and GLUT1 mRNA was quantified using qRT-PCR. D, RCC4-MOCK or RCC4-VHL cells were grown for 48 hours in normoxia (21% O2) or hypoxia (1% O2) for 48 hours, then lysed and immunoblotted with the indicated antibodies. E, RCC4-MOCK cells were transfected with siRNA directed to HIF1α or HIF2α. mRNA expression of GAL3ST1 was quantified. Protein expression of HIF1α or HIF2α was quantified to ensure efficient knockdown was achieved. F, The GAL3ST1 promoter sequence containing two putative HREs (HRE1 and HRE2). HRE1 is approximately 800 bp upstream of HRE2. G, HEK293A cells were transfected with (+) or without (−) HA-HIF1α (P>A) and/or HA-HIF2α (P>A) in combination with the indicated luciferase vectors. Cells were lysed and a luciferase assay was performed; luciferase activity was normalized to protein content (RLU/μg). Lysates were also immunoblotted with the indicated antibodies to verify equivalent HA-HIFα (P>A) expression. Western blots are aligned with the line graph below to signify the particular luciferase construct coexpressed with MOCK and HA-HIFα (P>A) plasmids. Experiments were performed in triplicate. H, RCC4-VHL cells were cultured in either normoxia (21% O2) or hypoxia (1% O2) and a ChIP was performed using either anti-HIF1α antibody or anti-HIF2α antibody. Genomic DNA fragments comprised of the 5′-UTR region of GAL3ST1 [GAL3ST1 (HRE2)] and a control region internal to GAL3ST1 [GAL3ST1 (internal)], and the β-ACTIN promoter were quantified in the immunoprecipitated samples by qRT-PCR and normalized to total genomic input signals (% input). All experiments were analyzed using an unpaired t test with a 95% confidence interval. *, P < 0.05; **, P < 0.01; ***, P < 0.001. All experiments were performed three times and error bars represent SDs of the mean. RLU, raw light units; IP, immunoprecipitate.
- Figure 3.
GAL3ST1 expression leads to increased levels of sulfatides in the cell membrane of ccRCC cells. A, Depiction of the pathway that results in the synthesis of sulfatides (SM3 and SM4) and is catalyzed by GAL3ST1, also called CST. B, 786-MOCK and 786-VHL cells were pooled, as were RCC4-MOCK and RCC4-VHL cells. Cells were cultured under normoxia (21% O2) or hypoxia (1% O2) for 48 hours, fixed, and immunofluorescently stained for sulfatides (green), HA-VHL (red), and DAPI nuclear stain. Exposure times were automatically optimized by the imaging software. C, 786 and RCC4-shSCR and -shGAL3ST1 cells were fixed and immunofluorescently stained for sulfatide (red) and the nuclear stain DAPI (blue). D, 786 and RCC4-shSCR and -shGAL3ST1 cells were lysed and protein expression of GAL3ST1 was analyzed using Western blot. β-actin was used as a loading control.
- Figure 4.
Sulfatides promote platelet binding to cancer cells. A, Wells of a 96-well plate were coated with 2 μg/mL BSA or bovine sulfatides. Washed human platelets from healthy donors were stained with calcein AM and incubated in the coated wells. Following washes, florescence was measured using a fluorescent plate reader. Results are presented as a percent change in fluorescence of control (BSA). Data were analyzed using unpaired Student t test where, ***, P < 0.001. B, 786-shSCR and 786-shGAL were coincubated with human platelets from healthy donors stained with wheat germ agglutinin (red). Actin (green) stained both cells and activated platelets, while nuclei were stained with Hoechst (blue). C, An example of flow cytometry data acquired demonstrating the emergence of a double-positive cell population. Platelets were coincubated with GFP+ renal cancer cells for 5 minutes. Samples were fixed, stained with PE-CD41, and analyzed using flow cytometry. D, The percentage of a GFP+ PE-CD41+ population was graphed for each platelet donor tested (n = 3).
- Figure 5.
Platelets protect GAL3ST1-sulfatide–expressing cells from NK cell–mediated destruction but not GAL3ST1-sulfatide–negative cells. NK-92 cells were stained with Calcein-Red. 786-shSCR or -shGAL3ST1 are GFP+. Cancer cells were incubated for 3 hours in the presence or absence of NK-92 cells ± washed healthy platelets. After incubations, cells were stained for 7-AAD and Annexin V and cell death was quantified using flow cytometry. A, An example of the gating regime applied to the experiments presented and the significant shift in AnnexinV+ cells upon coincubation of ccRCC cells with NK-92 cells. A total of 10,000 events were recorded for each experiment. B, Cell death in 786-shSCR and 786-shGAL3ST1 cells coincubated in the presence or absence of platelets (P) or NK-92 cells (NK). Untreated cancer cells are presented in the figure as NT. n = 5 with washed human platelets from 5 healthy donors. C, NK-mediated cell death in samples coincubated with platelets in 786-shSCR and 786-shGAL3ST1 cells. A paired t test was used to evaluate significance between the groups where n = 5 healthy donors. D, Using the same experimental setup, platelets were activated with U46619 and then coincubated with shSCR or shGAL3ST1 ± NK-92 cells. In figures B and D, a one-way ANOVA with post hoc Tukey test was applied to determine statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant).
- Figure 6.
Model of GAL3ST1 in NK cell–mediated tumor cell killing. VHL−/− renal cancer cells express high levels of GAL3ST1 and NK cells can target these cells for destruction (left). In the presence of platelets that interact with VHL−/− cells, NK cells have significantly restricted abilities to target renal cancer cells for destruction (middle). When GAL3ST1 expression is decreased, platelet interactions with renal cancer cells are perturbed and NK-mediated killing is increased (right).
Tables
- Table 1.
A list of the most upregulated genes in 10 ccRCC samples compared with normal kidney
Rank Gene ID Gene symbol 1 54901 NDUFA4L2 2 84634 KISS1R 3 7130 TNFAIP6 4 4885 NPTX2 5 1573 CYP2J2 6 2173 FABP7 7 51129 ANGPTL4 8 112399 EGLN3 9 100133188 NA 10 5169 ENPP3 ..... ..... ..... 42 1520 CTSS 43 9514 GAL3ST1 44 714 C1QC NOTE: Affymetrix microarray expression analysis of 10 primary ccRCC tumor samples. Analyzed values for each gene were averaged across all 10 samples and the averages were ranked from highest to lowest.
Supplementary Data
- Supplementary Data - Microarray expression data
- Supplementary Figure 1 - S1. ChIP Atlas was used to interrogate HIF1, EPAS and ARNT binding at the GAL3ST1 locus using ChIP-seq data from various kidney (cancerous and healthy) cells
- Supplementary Figure 2 - S2. RCC4 ChIP -seq data (GEO accession #GSE120885) for HIF1 and HIF2 at GAL3ST1 promoter, including regions 'HRE1' and 'HRE2'.
Volume 17, Issue 11
