The Sda Synthase B4GALNT2 Reduces Malignancy and Stemness in Colon Cancer Cell Lines Independently of Sialyl Lewis X Inhibition

Abstract

Background: The Sd^a antigen and its biosynthetic enzyme B4GALNT2 are highly expressed in healthy colon but undergo a variable down-regulation in colon cancer. The biosynthesis of the malignancy-associated sialyl Lewis x (sLe^x) antigen in normal and cancerous colon is mediated by fucosyltransferase 6 (FUT6) and is mutually exclusive from that of Sd^a. It is thought that the reduced malignancy associated with high B4GALNT2 was due to sLe^x inhibition.

Methods: We transfected the cell lines SW480 and SW620, derived respectively from a primary tumor and a metastasis of the same patient, with the cDNAs of FUT6 or B4GALNT2, generating cell variants expressing either the sLe^x or the Sd^a antigens. Transfectants were analyzed for growth in poor adherence, wound healing, stemness and gene expression profile.

Results: B4GALNT2/Sd^a expression down-regulated all malignancy-associated phenotypes in SW620 but only those associated with stemness in SW480. FUT6/sLe^x enhanced some malignancy-associated phenotypes in SW620, but had little effect in SW480. The impact on the transcriptome was stronger for FUT6 than for B4GALNT2 and only partially overlapping between SW480 and SW620.

Conclusions: B4GALNT2/Sd^a inhibits the stemness-associated malignant phenotype, independently of sLe^x inhibition. The impact of glycosyltransferases on the phenotype and the transcriptome is highly cell-line specific.

Keywords: Sda antigen; cancer stem cells; non-adherent growth; sialyl Lewis antigens; transcriptomic analysis.

Figure 1

The Sd^a and the sLe^x antigens derive from alternative and mutually exclusive terminations of a common α2,3-sialylated type 2 structure. The key enzymes for their biosynthesis in colonic tissues are FUT6 and B4GALNT2.

Figure 2

Phenotypic effects induced by FUT6 or B4GALNT2 expression. (A) The doubling time (DT), expressed in hours, was calculated as described in Materials and Methods in at least three independent experiments performed in duplicate. (B) Fifty cells of the different populations were seeded in a 6-well plate in standard conditions of growth. After 15 days, the colonies were fixed, stained and counted. Histograms indicate the total number of colonies. The assay was repeated 8 times. (C) Ten thousand cells of the different populations were seeded in 6-well plates in 0.33% agar. After 3 weeks, the colonies were fixed, stained, photographed without magnification and the colonies visible to the naked-eye were counted. (D) Each well of a 6-well plate was coated with a 0.5% agar solution in complete L-15 medium to avoid adhesion of the cells to the plastic surface. Ten thousand cells were then seeded in complete L-15 medium. The aspect of the spheroids is shown, but the photographs are not necessarily representative of their amount. To quantify the number of cells grown as spheroids or single cells in liquid phase, the cells were quantitatively collected, pelleted by centrifugation and homogenized. The total amount of protein was calculated and taken as a measure of the cells grown. (E) The healing of a wound in a monolayer of confluent cells was monitored every 24 h. The free area of the wound was quantitated by ImageJ and normalized to the free area of the same cell line at 0 h, which was taken as 100%. The photographs show only the start (0 h) and the end point (96 h) of the healing process. Graphs in the bottom report the quantification of the healing process at each time point. The microphotographs were taken at a 4× magnification. In (A–E) statistical analysis was performed using one-way analysis of variance (ANOVA) and Dunnett’s multiple comparisons test. ns, not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Figure 3

ALDEFLUOR analysis. Cells were incubated with ALDEFLUOR either in the presence or in the absence of the inhibitor N,N-diethylaminobenzaldehyde (DEAB). Gates excluding all of the cells labelled in the presence of DEAB were set. Cells included in the gate in the absence of DEAB, were considered to be aldheyde dehydrogenase 1 (ALDH)-positive. Histograms report the percentage of ALDH positive cells ±SD in five independent experiments. * p ≤ 0.05.

Figure 4

Heatmaps of genes modulated upon FUT6 and B4GALNT2 expression. (A) Cluster analysis of SW480 and SW620 cells transfected with FUT6 and compared with control Neo. (B) Cluster analysis of SW480 and SW620 cells transfected with B4GALNT2 and compared with control Neo. Differentially expressed genes are reported. Genes (columns) and samples (rows) were grouped by hierarchical clustering (Manhattan correlation). High- and low-expression was normalized to the average expression across all samples. Differences were analyzed by the moderated t-test. Corrected p-value cut-off: 0.05; multiple test correction used: Benjamini–Hochberg. Color codes refer to the level of up- or down-regulation.