C2-O-sLeX Glycoproteins Are E-Selectin Ligands that Regulate Invasion of Human Colon and Hepatic Carcinoma Cells

Similar to mechanisms of recruitment of activated leukocytes to inflamed tissues, selectins mediate adhesion and extravasation of circulating cancer cells. Our objective was to determine whether sialyl Lewis X modified core 2 O-glycans (C2-O-sLeX) present on colon and hepatic carcinoma cells promote their adhesion and invasion. We examined membrane expression of C2-O-sLeX, selectin binding, invasion of human colon and hepatic carcinoma cell lines, and mRNA levels of alpha-2,3 fucosyltransferase (FucT-III) and core 2 beta-1,6 N-acetylglucosaminyltransferase (C2GnT1) genes, necessary for C2-O-sLeX synthesis, by quantitative reverse-transcriptase (RT) PCR. Synthesis of core 2 branched O-glycans decorated by sLeX is dependent on C2GnT1 function and thus we determined enzyme activity of C2GnT1. The cell lines that expressed C2GnT1 and FucT-III mRNA by quantitative RT-PCR were highly positive for C2-O-sLeX by flow cytometry, and colon carcinoma cells possessed highly active C2GnT1 enzyme. Cells bound avidly to E-selection but not to P- and L-selectin. Gene knock-down of C2GnT1 in colon and hepatic carcinoma cells using short hairpin RNAs (shRNA) resulted in a 40–90% decrease in C2-O-sLeX and a 30–50% decrease in E-selectin binding compared to control cells. Invasion of hepatic and colon carcinoma cells containing C2GnT1 shRNA was significantly reduced compared to control cells in Matrigel assays and C2GnT1 activity was down-regulated in the latter cells. The sLeX epitope was predominantly distributed on core 2 O-glycans on colon and hepatic carcinoma cells. Our findings indicate that C2GnT1 gene expression and the resulting C2-O-sLeX carbohydrates produced mediate the adhesive and invasive behaviors of human carcinomas which may influence their metastatic potential

C2GnT1 gene knockdown decreases C2-O-sLe^X glycoproteins and C2GnT1 activity.

<p>(A) C2GnT1 suppression by shRNA resulted in decreased mRNA levels of C2GnT1 but not FucT-III in LS174T cells, (scrambled shRNA vector, lane 1, and C2GnT1 shRNA vector, lane 2). (B) Similar effects on C2GnT1 mRNA but not FucT-III mRNA were observed in HepG2 cells after transient transfection with C2GnT1 shRNA (empty control vector, lane 1, and targeting shRNA vector, lane 2). Densitometry values are normalized to β-actin. (C) The C2GnT1 glycoprotein (50 kDa) was present in scrambled shRNA transduced LS174T cells (lane 1) but absent in C2GnT1 shRNA transduced cells (lane 2) as detected by immunoprecipitation and Western blotting using a polyclonal anti-C2GnT1 antibody. (D) The procedures were repeated using the C2GnT1 antibody for immunoprecipitation and a negative control IgG antibody for Western blotting. Note the absence of a detected C2GnT1 protein 50 kDa band. (D) We observed a 30–40% decrease in reactivity with CHO-131 mAb by flow cytometry for LS174T and HepG2 cells that contained C2GnT1 shRNA. Representative data from multiple experiments are shown. (E) C2GnT1 enzyme activity, measured as nmols of N-acetylglucosamine (GlcNAc) transferred per mg protein per hour in a glycosyltransferase assay, was also significantly decreased in C2GnT1 shRNA transduced LS174T cells, *p = 0.02. Mean values ± standard deviations of two independent experiments are presented.</p

C2GnT1 and FucT-III genes are endogenously expressed in human carcinoma cell lines.

<p>(A) Endogenous C2GnT1 and FucT-III mRNA transcripts in LS174T colorectal adenocarcinoma (lane 1) and HepG2 hepatic carcinoma (lane 2) cell lines were detected by RT-PCR. (B) Flow cytometric analysis of positive staining of LS174T and HepG2 cells labeled with CHO-131 mAb (anti-C2-O-sLe^X). A representative example of 3 experiments is shown for (A-B). (C) LS174T (*p = 0.008) and HepG2 cells (#p = 0.001) substantially bind to E-selectin in the presence of calcium ions (Ca²⁺). Binding is impeded in the presence of EDTA, a calcium ion chelator. The average of two experiments is shown.</p

C2GnT1 mediates invasion.

<p>(A) LS174T cells transduced with C2GnT1 shRNA were significantly less invasive than cells transduced with scrambled shRNA, *p<0.0001, in Matrigel transwell invasion assays. (B) Similarly, decreased invasion was observed for HepG2 cells that were transfected with C2GnT1 shRNA compared to transfection with a control vector, *p = 0.0005. (C) Representative photomicrographs from two separate experiments of invasion of LS174T cells transduced with the scrambled shRNA vector compared to (D) cells transduced with C2GnT1 shRNA and (E) HepG2 cells transfected with the control vector compared to (F) HepG2 cells transfected with C2GnT1 shRNA. The invading cells were stained and counted in five separate fields of view at 100X magnification. Arrows indicate invasive cells.</p

C2GnT1 gene knock-down results in decreased binding of cells to E-selectin.

<p>(A) Significant differences in E-selectin binding were not observed among LS174T un-manipulated cells, cells transduced with scrambled shRNA, or with C2GnT1 shRNA by flow cytometry. (B) E-selectin binding was significantly decreased for HepG2 cells transfected with C2GnT1 shRNA compared to cells transfected with the control vector, *p = 0.02. (C) Shear flow assays were performed at an E-selectin/Fc chimera concentration of 1 µg/ml and shear stresses ranging from 0.5 – 1.5 dynes/cm² in the presence of 10 µg/ml of an IgG isotype control mAb. At a shear stress of 0.5 dynes/cm², significantly fewer C2GnT1 shRNA transduced cells accumulated on E-selectin than un-manipulated LS174T cells, *p = 0.04. (D) In the same run of experiments, at a shear stress of 0.5 dynes/cm², significantly fewer un-manipulated LS174T cells treated with a 10 µg/ml of a blocking anti-E-selectin mAb accumulated on E-selectin than those cells treated with an isotype control mAb, ^#p = 0.004. Significantly fewer LS174T cells transduced with C2GnT1 shRNA and treated with a blocking anti-E-selectin mAb accumulated on E-selectin than those cells treated with an isotype control mAb, ^&p = 0.01. The same assay as shown in (C) at 0.5 dynes/cm² is included for comparison. For both groups of cells treated with an isotype control mAb, significantly fewer un-manipulated LS174T cells accumulated on E-selectin than LS174T cells transduced with C2GnT1 shRNA, *p = 0.04. Each continuous shear flow assay was performed in duplicate for each shear stress and the bars represent the mean ± standard deviations. Representative data from three independent experiments are shown.</p

Pathway of C2-O-sLe^X biosynthesis.

<p>A simplified version of C2-O-sLe^X biosynthesis is shown. Core 1 O-glycans are synthesized by addition of β1,3 galactose to N-acetylgalactosamine. The core 2 β1,6-<i>N</i>-acetylglucosaminyltransferase (C2GnT1) enzyme catalyzes the addition of a β1,6 branch to a core 1 O-glycan to form a core 2 β1,6 branched O-glycan. The core 2 O-glycan structure is further modified by a series of enzymatic reactions (omitted for clarity) including modification by α2,3 sialyltransferase and α1,3 fucosyltransferase (FucT-III) to form a sLe^X terminus (dotted box). These modifications result in the synthesis of the sLe^X-modified core 2 β1,6 O-glycan (C2-O-sLe^X) structure. GalNAc, N-acetylgalactosamine; Gal, galactose; GlcNAc, N-acetylglucosamine; NeuAc, sialic acid; Fuc, fucose.</p