HCELL Is the Major E- and L-selectin Ligand Expressed on LS174T Colon Carcinoma Cells

Monica M Burdick,Julia T Chu,Robert Sackstein,Samuel Godar

doi:10.1074/jbc.m513617200

Abstract

Engagement of vascular E-selectin and leukocyte L-selectin with relevant counter-receptors expressed on tumor cells contributes to the hematogenous spread of colon carcinoma. We recently demonstrated that the LS174T colon carcinoma cell line expresses the CD44 glycoform known as hematopoietic cell E-/L-selectin ligand (HCELL), which functions as a high affinity E- and L-selectin ligand on these cells. To define the contribution of HCELL to selectin-mediated adhesion on intact tumor cells, we measured the binding of LS174T cells transduced with CD44 short interfering RNA (siRNA) or with vector alone to 6-h interleukin-1beta-stimulated human umbilical vein endothelial cells (HUVEC) and to human peripheral blood mononuclear cells (PBMC) under physiological flow conditions. LS174T cell attachment to HUVEC was entirely E-selectin-dependent, and PBMC tethering to tumor cell monolayers was completely L-selectin-dependent. At physiological shear stress, CD44 siRNA transduction led to an approximately 50% decrease in the number of LS174T cells binding to stimulated HUVEC relative to vector alone-transduced cells. CD44 siRNA-transduced cells also rolled significantly faster than vector-transduced cells on HUVEC, indicating prominent HCELL participation in stabilizing tumor cell-endothelial adhesive interactions against fluid shear. Furthermore, HCELL was identified as the principal L-selectin ligand on LS174T cells, as PBMC binding to CD44 siRNA-transduced tumor cells was reduced approximately 80% relative to vector-transduced cells. These data indicate that expression of HCELL confers robust and predominant tumor cell binding to E- and L-selectin, highlighting a central role for HCELL in promoting shear-resistant adhesive interactions essential for hematogenous cancer dissemination.

Highlights

When confined to a primary site, the five-year survival rate from colon cancer is ϳ90%
E-/L-selectin Ligand Activity of hematopoietic cell E-/L-selectin ligand (HCELL) on Colon Cancer binding strength) and a nearly complete (Ͼ80%) loss in L-selectin ligand activity over a physiologically relevant shear stress range. These findings provide compelling evidence that HCELL is the predominant E- and L-selectin glycoprotein ligand expressed on LS174T colon carcinoma cells and suggest that therapeutic strategies to inhibit HCELL expression/function on cancer cells bearing this CD44 glycoform may be beneficial in the prevention of metastasis
We recently identified and characterized the sialofucosylated HCELL modification on primarily high molecular weight vCD44 isoform(s) of the LS174T colon carcinoma cell line and demonstrated its function as a high affinity E- and L-selectin ligand using the blot rolling assay (17, 18)

Summary

EXPERIMENTAL PROCEDURES

Antibodies, and Reagents—LS174T human colon adenocarcinoma cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured in the recommended medium (minimal essential medium containing 10% FBS, 1% sodium-pyruvate, 1% nonessential amino acids, and 1% penicillin/streptomycin). For LS174T cell interactions with HUVEC, attachment assays were performed by perfusing cells (106/ml in Hanks’ balanced salt solution containing 10 mM HEPES, 2 mM CaCl2 (H/H/ Ca2ϩ), and 5% FBS) at the appropriate flow rates to obtain wall shear stresses of 1.0 –2.0 dynes/cm, thereby mimicking the fluid mechanical environment of the post-capillary venules (22). PBMC in 5% FBS/H/H/Ca2ϩ were perfused at 2 ϫ 106/ml at shear stresses from 0.2 to 2.0 dynes/cm, and the number of PBMC interacting with LS174T monolayers in four independent fields of view after a 2-min attachment period was evaluated. Nonspecific adhesion was assessed by perfusing CHO-E cell suspensions containing function-blocking anti-Eselectin mAb, using 5 mM EDTA in the flow medium or by perfusing mock transfectants (CHO-mock)

RESULTS

Shear Stress

DISCUSSION