Relationship of sialyl-Lewis(x/a) underexpression and E-cadherin overexpression in the lymphovascular embolus of inflammatory breast carcinoma

Abstract

Inflammatory breast carcinoma (IBC) is characterized by florid tumor emboli within lymphovascular spaces called lymphovascular invasion. These emboli have a unique microscopic appearance of compact clumps of tumor cells retracted away from the surrounding endothelial cell layer. Using a human SCID model of IBC (MARY-X), we, in previous studies, demonstrated that the tumor cell embolus (IBC spheroid) forms on the basis of an intact and overexpressed E-cadherin/alpha,beta-catenin axis that mediates tumor cell-tumor cell adhesion. In the present study we examine the mechanism behind the apparent lack of binding of the tumor embolus to the surrounding endothelium. We find that this lack of tumor cell binding is because of markedly decreased sialyl-Lewis(x/a) (sLe(x/a)) carbohydrate ligand-binding epitopes on its overexpressed MUC1 and other surface molecules that bind endothelial E-selectin. Decreased sLe(x/a) is because of decreased alpha3/4-fucosyltransferase activity in MARY-X. The decreased sLe(x/a) fail to confer electrostatic repulsions between tumor cells, which further contributes to the compactness of the MARY-X spheroid by allowing the E-cadherin homodimeric interactions to go unopposed. MARY-X spheroids were retrovirally transfected with FucT-III cDNA, significantly raising their levels of fucosyltransferase activity and surface sLe(x/a). In parallel experiments, enzymatic transfers with a milk alpha1,3-fucosyltransferase and an alpha2,3-sialyltransferase (ST3GalIV) were performed on the MARY-X spheroids and increased surface sLe(x/a). The addition of sLe(x/a) by either manipulation caused disadherence of the MARY-X spheroids and the disruption of the E-cadherin homodimers mediating cell adhesion. Our findings support the cooperative relationship of sLe(x/a) underexpression and E-cadherin overexpression in the genesis of the lymphovascular embolus of IBC.

Figure 1.

A: Lymphovascular invasion in IBC in humans is characterized by numerous tumor emboli in lymphovascular spaces. B: The presence of tumor cell-tumor cell homotypic adhesion and the lack of tumor cell-endothelial cell heterotypic adhesion is evidenced by this lymphovascular tumor embolus of MARY-X by light microscopy. H&E stain; original magnifications: ×250 (A); ×400 (B).

Figure 2.

A: Spheroids of MARY-X form tight clusters in which individual cells cannot be distinguished. B: Although this compactness is mediated by E-cadherin, these spheroids also demonstrate MUC1 immunoreactivity. A, Phase contrast (original magnification, ×200); B, anti-MUC1, immunoperoxidase.

Figure 3.

A: Like Colo-205, MARY-X spheroids overexpress MUC1 but unlike Colo-205, MARY-X exhibits markedly decreased levels of sLe^x and sLe^a by Western blot (B). Levels of sLe^x and sLe^a can be increased in MARY-X through either exogenous enzymatic fucosylation (sLe^x/a-MARY-X) or FucT-III transfection (FucT-III-MARY-X). A, Western blot, anti-MUC1; B, Western blot, anti-sLe^x and anti-sLe^a.

Figure 4.

Comparative assay of α3/4-fucosyltransferase activities in wild-type MARY-X, Colo-201, Colo-205, and FucT-III-MARY-X reveals low endogenous levels of both fucosyltransferases in wild-type MARY-X and increased levels after FucT-III transfection. H-type-I (Galβ1–3GlcNAc) is the oligosaccharide acceptor for α4-fucosyltransferase activity and H-type-2 (Galβ1–4GlcNAc) is the oligosaccharide acceptor for α3-fucosyltransferase activity.

Figure 5.

A: FucT-III-MARY-X are 60 μm in size when transfected and enlarge to 80 μm in size at 72 hours (B). SLe^x/a-MARY-X exhibits a similar enlargement. Subsequent FucT-III-MARY-X (C) and sLe^x/a-MARY-X (D) disadherence occurs with the former being complete and the latter being partial. E: Disadhering FucT-III-MARY-X exhibits strong sLe^x surface immunoreactivity and persistence of E-cadherin immunoreactivity even when fully disadhered (F). Original magnifications: ×200 (A–D, phase contrast); ×200 (E, anti-sLe^x, immunoperoxidase); ×100 (F, anti-E-cadherin, immunoperoxidase).

Figure 6.

A: Disadherence produced profound apoptosis by terminal dUTP nick-end labeling. B: Different disadherence mechanisms all increase AI, suggesting a common apoptosis mechanism. C: Attachment of Colo-205, Colo-201, and different MARY-X spheroids to stimulated and unstimulated HUVECs is depicted. D: Adhesion of these same cell types to wells coated with E-selectin is depicted. Attachment (C) and adhesion (D) assays of tumor cells and/or spheroids as detailed in Materials and Methods. Attachment and adhesion are quantitated by counting cells and/or spheroids within a 0.1-mm² grid. Each bar represents the mean cell count ± SD of four to eight wells.

Figure 7.

Schematic depicts hypothesis of homotypic E-cadherin interactions and lack of heterotypic MUC1/sLe^x/a-E-selectin interactions being cooperative in nature.