A Novel Function of CD82/KAI1 in Sialyl Lewis Antigen-Mediated Adhesion of Cancer Cells: Evidence for an Anti-Metastasis Effect by Down-Regulation of Sialyl Lewis Antigens

We have recently elucidated a novel function for CD82 in E-cadherin-mediated homocellular adhesion; due to this function, it can inhibit cancer cell dissociation from the primary cancer nest and limit metastasis. However, the effect of CD82 on selectin ligand-mediated heterocellular adhesion has not yet been elucidated. In this study, we focused on the effects of the metastasis suppressor CD82/KAI1 on heterocellular adhesion of cancer cells to the endothelium of blood vessels in order to further elucidate the function of tetraspanins. The over-expression of CD82 in cancer cells led to the inhibition of experimentally induced lung metastases in mice and significantly inhibited the adhesion of these cells to human umbilical vein epithelial cells (HUVECs) in vitro. Pre-treatment of the cells with function-perturbing antibodies against sLe^a/x significantly inhibited the adhesion of CD82-negative cells to HUVECs. In addition, cells over-expressing CD82 exhibited reduced expression of sLe^a/x compared to CD82-negative wild-type cells. Significant down-regulation of ST3 β-galactoside α-2, 3-sialyltransferase 4 (ST3GAL4) was detected by cDNA microarray, real-time PCR, and western blotting analyses. Knockdown of ST3GAL4 on CD82-negative wild-type cells inhibited expression of sLe^x and reduced cell adhesion to HUVECs. We concluded that CD82 decreases sLe^a/x expression via the down-regulation of ST3GAL4 expression and thereby reduces the adhesion of cancer cells to blood vessels, which results in inhibition of metastasis.     

Sialyl Lewis-dependent binding of human monocyte-derived dendritic cells to selectins

The limited efficacy of monocyte-derived dendritic cell (mo-DC)-based vaccines is primarily attributed to the reduced mo-DC migratory capacity. One undefined aspect is the initial binding of mo-DCs to endothelial cells and vascular selectins. In this study, we investigated the role and modulation of the selectin binding determinant sialyl Lewis^x (sLe^x) in selectin-dependent mo-DC binding. Our data reveal that sLe^x is required for maximal binding of mo-DCs to tumor necrosis factor (TNF)-α-activated endothelial cells under static conditions, as evidenced by the use of sialidase. Sialidase treatment also abrogated mo-DC cell tethering to immobilized, purified P-, L-, or E-selectin under flow. The requirement of sLe^x-dependent binding of mo-DC to selectins was further substantiated by using sLe^x free sugar and anti-sLe^x antibody, which significantly suppressed mo-DC-selectin binding. P-selectin glycoprotein ligand-1 is required for mo-DC binding to both P- and L-selectin, but it is dispensable for E-selectin recognition. Interestingly, the extent of mo-DC tethering was maximal on P-selectin, followed by E- and L- selectin. Accordingly, L-selectin mediated faster mo-DC rolling than E- or P-selectin. Interferon (IFN)-γ induces a significant increase in mo-DC surface sLe^x expression, which is probably due to the enhanced synthesis of C2GnT-I. These findings may contribute to improving mo-DC-based vaccination protocols.     

Molecular Plasticity of E-Cadherin and Sialyl Lewis X Expression,in Two Comparative Models of Mammary Tumorigenesis

Background

The process of metastasis involves a series of steps and interactions between the tumor embolus and the microenvironment. Key alterations in adhesion molecules are known to dictate progression from the invasive to malignant phenotype followed by colonization at a distant site. The invasive phenotype results from the loss of expression of the E-cadherin adhesion molecule, whereas the malignant phenotype is associated with an increased expression of the carbohydrate ligand-binding epitopes, (e.g. Sialyl Lewis ^x/a) that bind endothelial E-selectin of the lymphatics and vasculature.

Methodology

Our study analyzed the expression of two adhesion molecules, E-cadherin and Sialyl Lewis x (sLe^x), in both a canine mammary carcinoma and human inflammatory breast cancer (IBC) model, using double labelled immunofluorescence staining.

Results

Our results demonstrate that canine mammary carcinoma and human IBC exhibit an inversely correlated cellular expression of E-cadherin and sLe^x within the same tumor embolus.

Conclusions

Our results in these two comparative models (canine and human) suggest the existence of a biologically coordinated mechanism of E-cadherin and sLe^x expression (i.e. molecular plasticity) essential for tumor establishment and metastatic progression.     

Cell-surface nucleolin is a signal transducing P-selectin binding protein for human colon carcinoma cells

We have previously shown that P-selectin binding to Colo-320 human colon carcinoma cells induces specific activation of the alpha(5)beta(1) integrin with a concomitant increase of cell adhesion and spreading on fibronectin substrates in a phosphatidylinositol 3-kinase (PI3-K) and p38 MAPK-dependent manner. Here, we identified by affinity chromatography and characterized nucleolin as a P-selectin receptor on Colo-320 cells. Nucleolin mAb D3 significantly decreases the Colo-320 cell adhesion to immobilized P-selectin-IgG-Fc. Moreover, nucleolin becomes clustered at the external side of the plasma membrane of living, intact cells when bound to cross-linked P-selectin-IgG-Fc chimeric protein. We have also found P-selectin binding to Colo-320 cells induces tyrosine phosphorylation specifically of cell-surface nucleolin and formation of a signaling complex containing cell-surface nucleolin, PI3-K and p38 MAPK. Using siRNA approaches, we have found that both P-selectin binding to Colo-320 cells and formation of the P-selectin-mediated p38 MAPK/PI3-K signaling complex require nucleolin expression. These results show that nucleolin (or a nucleolin-like protein) is a signaling receptor for P-selectin on Colo-320 cells and suggest a mechanism for linkage of nucleolin to P-selectin-induced signal transduction pathways that regulate the adhesion and the spreading of Colo-320 on fibronectin substrates.     

Expression of ST3GAL4 Leads to SLex Expression and Induces c-Met Activation and an Invasive Phenotype in Gastric Carcinoma Cells

Sialyl-Lewis X (SLe^x) is a sialylated glycan antigen expressed on the cell surface during malignant cell transformation and is associated with cancer progression and poor prognosis. The increased expression of sialylated glycans is associated with alterations in the expression of sialyltransferases (STs). In this study we determined the capacity of ST3GAL3 and ST3GAL4 sialyltransferases to synthesize the SLe^x antigen in MKN45 gastric carcinoma cells and evaluated the effect of SLe^x overexpression in cancer cell behavior both in vitro and in vivo using the chicken chorioallantoic membrane (CAM) model. The activation of tyrosine kinase receptors and their downstream molecular targets was also addressed. Our results showed that the expression of ST3GAL4 in MKN45 gastric cancer cells leads to the synthesis of SLe^x antigens and to an increased invasive phenotype both in vitro and in the in vivo CAM model. Analysis of phosphorylation of tyrosine kinase receptors showed a specific increase in c-Met activation. The characterization of downstream molecular targets of c-Met activation, involved in the invasive phenotype, revealed increased phosphorylation of FAK and Src proteins and activation of Cdc42, Rac1 and RhoA GTPases. Inhibition of c-Met and Src activation abolished the observed increased cell invasive phenotype. In conclusion, the expression of ST3GAL4 leads to SLe^x antigen expression in gastric cancer cells which in turn induces an increased invasive phenotype through the activation of c-Met, in association with Src, FAK and Cdc42, Rac1 and RhoA GTPases activation.     

Expression of the blood-group-related antigens Sialyl Lewis a,Sialyl Lewis x and Lewis y in term placentas of normal,preeclampsia, IUGR- and HELLP-complicated pregnancies

Lewis antigens belong to the blood group of antigens and mediate cellular adhesion through interaction with selectins. Invasive trophoblasts use an array of adhesion molecules to facilitate cell–cell and cell–extracellular matrix interactions. Here, we examined immunohistochemically the expression of Sialyl Lewis a (sLe^a), Sialyl Lewis x (sLe^x) and Lewis y (Le^y) in term placentas obtained from cases of normal, intrauterine growth retardation (IUGR), preeclamptic (PE) and hemolysis, elevated liver enzymes and low platelets syndrome (HELLP) pregnancies. We report the expression of sLe^x in third trimester extravillous trophoblasts (EVT). sLe^x was significantly decreased in IUGR and moderately decreased in PE compared to normal placentas. sLe^x was additionally found in syncytiotrophoblast, without however any significant differences in staining intensity between normal and pathological cases. sLe^a was restricted to amnion epithelium. Finally, Le^y was expressed in cytotrophoblasts and villous endothelial cells. Le^y expression was significantly upregulated in IUGR and HELLP, whereas there was a trend toward increase in PE compared to normal placentas. The present study suggests that downregulation of sLe^x in EVT might be associated with IUGR and PE. Furthermore, Le^y, which was recently described as a potent angiogenic factor, is upregulated in placental villi in conditions associated with placental malperfusion. U. Jeschke and A. Makrigiannakis have contributed equally.     

Detection in human blood platelets of sialyl Lewis X gangliosides, potential ligands for CD62 and other selectins

Activated platelets are known to express P-selectin, a lectin-likeadhesion receptor (CD62), through which they bind to sialylLewis X (sLe^x) ligands displayed on the membranes of leukocytes.To determine whether direct platelet-platelet interactions viaP-selectin/sLe^x interactions are also possible, we have examinedthe ganglioside extract of human blood platelets for the presenceof sLe^x ligands. Using the sensitive method of high-performancethin-layer chromatography (HPTLC)-immunostaining with the monoclonalantibody (mAb) CSLEX or with sialidase followed by mAbs MC480or PM81, eight sLe^x bands were demonstrated at R₁ 0.01, 0.03,0.05, 0.06, 0.08, 0.10, 0.14 and 0.21 in the solvent 45:55:10chloroform-methanol-aqueous 0.02% CaCl₂. The sensitivity ofall eight bands to sialidase or endoglycoceramidase confirmedthat they were gangliosides. Comparison of the HPTLC mobilitiesand densities of platelet bands with those from five other humantissues (granulocytes, monoblasts, kidney, aortic endotheliumand erythrocytes) in three different solvents revealed threemajor bands associated with platelets: 3 (R₁ 0.03), 6 (0.08)and 14 (0.21). Platelet bands were demonstrated not to haveresulted from granulocyte contamination. Partial purificationof platelet sLe^x gangliosides by high-performance liquid chromatographyand their reaction with 14 oligosaccharide-specific mAbs (FH4,FH5, LM112-161, LM-181, A5, 1B2, BR55-2, BE2, ES4, MC631, MH04,SH34, P001 and MC813-70) revealed that band 6 is a multifucosylatedneolacto ganglioside and band 14 is a branched, disialo neolactofucoganglioside. Platelet band 3 combined the features of bothbands 6 and 14, and reacted differently than granulocyte band3. These partial structures resemble gangliosides associatedwith adhesion in other cell systems. It is concluded that plateletsexpress tissue-specific sLe^x gangliosides (sLe^x ligands). Thus,it is possible that platelet-platelet binding may be mediatedat least partially through P-selectin/sLe^x interactions, especiallyafter platelet activation. gangliosides HPTLC-immunostaining platelets selectin ligands sLe^x     

Critical role of mac-1 sialyl lewis x moieties in regulating neutrophil degranulation and transmigration

Leukocyte cell surface sialyl Lewis x (sLe^x) and related epitopes play an important role in cell rolling and adhesion during diapedesis via interaction with E-selectin. Here, we present evidence that Mac-1 (CD11b/CD18, CR-3) is a major neutrophil glycoprotein decorated with sLe^x and ligation of these carbohydrate moieties by anti-sLe^x antibody significantly impairs neutrophil functions. First, Western blot analysis shows that both CD11b and CD18 subunit of purified Mac-1 are decorated with sLe^x moieties. A significant co-localization of CD11b and sLe^x moieties is observed at neutrophil secondary granules. With stimulation of formyl-Met-Leu-Phe (fMLP), neutrophil surface labeling with anti-sLe^x antibody follows an identical up-regulation pattern of Mac-1. Second, protein-binding assays indicate that sLe^x moieties on Mac-1 are critical for binding interaction of Mac-1 to E-selectin. Removal of sLe^x moieties completely abolishes Mac-1-E-selectin binding. Finally, ligation of Mac-1 sLe^x by anti-sLe^x antibody induces a significant degranulation of neutrophil secondary granules at the absence of chemoattractant stimulation. This “dysregulated” degranulation induced by anti-sLe^x antibody strongly inhibits neutrophil transmigration in response to fMLP. In summary, Mac-1 sLe^x moieties play a critical role in regulating β₂ integrin functions during neutrophil transmigration and degranulation.     

Reduction of rat myocardial ischemia and reperfusion injury by sialyl Lewis x oligosaccharide and anti-rat P-selectin antibodies

Polymorphonuclear leukocytes (PMN) are directly involved indevelopment of ischemic myocardial injury. Adhesion of PMN toendothelial cells is an initial step that triggers a sequentialprocess leading to acute inflammatory responses. Interactionbetween P-selectin and its oligosaccharide ligand, sialyl Lewisx (sLe^x), plays an important role in the early stage of theadhesion. To examine the role of P-selectin in various animaldisease models especially in rats, we have cloned rat E- andP-selectin cDNAs and established monoclonal antibodies againstthese rat selectins. In this report, we describe the generationand characterization of anti-rat P-selectin antibodies (ARPs).These antibodies detect cell surface P-selectin on thrombin-stimulatedrat platelets. More importantly, intravenous administrationof ARP2-4 reduced infarction developed after 30 min of ischemiafollowed by 24 h of reperfusion in a rat myocardial injury model.In addition, similar protective effect was also observed byadministration of a sLe^x- oligosaccharide. These results indicatethat cell adhesion mediated via P-selectin is involved in thedevelopment of ischemia and reperfusion injury in rat heart. ischemia and reperfusion injury monoclonal antibodies selectins sialyl Lewis x     

Synthesis of sialyl Lewis<Superscript>a</Superscript> (sLe<Superscript>a</Superscript>, CA19-9) and construction of an immunogenic sLe<Superscript>a</Superscript> vaccine

Sialyl Lewis^a (sLe^a), also termed CA19-9 antigen, is recognized by murine mAb19-9 and is expressed on the cancer cell surface as a glycolipid and as an O-linked glycoprotein. It is highly expressed in a variety of gastrointestinal epithelial malignancies including colon cancer and pancreatic cancer, and in breast cancer and small cell lung cancer, but has a limited expression on normal tissues. sLe^a is known to be the ligand for endothelial cell selectins suggesting a role for sLe^a in cancer metastases and adhesion. For these reasons, sLe^a may be a good target for antibody mediated immunotherapy including monoclonal antibodies and tumor vaccines. However, sLe^a is structurally similar to sLe^x and other blood group related carbohydrates which are widely expressed on polymorphonucleocytes and other circulating cells, raising concern that immunization against sLe^a will induce antibodies reactive with these more widely expressed autoantigens. We have shown previously both in mice and in patients that conjugation of a variety of carbohydrate cancer antigen to keyhole limpet hemocyanin (KLH) and administration of this conjugate mixed with saponin adjuvants QS-21 or GPI-0100 are the most effective methods for induction of antibodies against these cancer antigens. We describe here for the first time the total synthesis of pentenyl glycoside of sLe^a hexasaccharide and its conjugation to KLH to construct a sLe^a-KLH conjugate. Groups of five mice were vaccinated subcutaneously four times over 6 weeks. Sera were tested against sLe^a-HSA by ELISA and against sLe^a positive human cell lines adenocarcinoma SW626 and small cell lung cancer (SCLC) DMS79 by FACS. As expected, mice immunized with unconjugated sLe^a plus GPI-0100 or unconjugated sLe^a mixed with KLH plus GPI-0100 failed to produce antibodies against sLe^a. However, mice immunized with sLe^a-KLH conjugate without GPI-0100 produced low levels of antibodies and mice immunized with sLe^a-KLH plus GPI-0100 produced significantly higher titer IgG and IgM antibodies against sLe^a by ELISA. These antibodies were highly reactive by FACS and mediated potent complement mediated cytotoxicity against sLe^a positive SW626 and DMS79 cells. They showed no detectable cross reactivity against a series of other blood group-related antigens, including Le^y, Le^x, and sLe^x by dot blot immune staining. This vaccine is ready for testing as an active immunotherapy for treating sLe^a positive cancer in clinical settings. Govind Ragupathi and Philip O. Livingston are paid consultants and shareholders in MabVax Therapeutics, Inc., San Diego, CA 92121. The sLe^a vaccine is licensed to MabVax.     

Sialyl Lewisx expression on IgG in rheumatoid arthritis and other arthritic conditions: a preliminary study

Both infiltrating leukocytes and soluble immunoglobulin form aggregates in synovial fluid during the inflammatory process in rheumatoid arthritis (RA). Some of these changes are probably mediated by the adhesion molecule, E-selectin, which increases its expression with disease activity. As glycosylation changes in IgG in RA are well established, the current study was undertaken to measure the expression of the carbohydrate antigen sialyl Lewis x (sLe^x), on IgG in RA. sLe^x is a major ligand for E-selectin. Using a recently developed ELISA, sLe^x expression was determined in IgG isolated from 8 healthy individuals, 20 RA sufferers (10 early and 10 with more long-standing disease) and 20 patients with other rheumatic conditions (osteoarthritis, ankylosing spondylitis, systemic lupus erythematosus). S Le^xexpression on IgG was elevated above the reference range in all but one of the RA patients and this change was highly significant (P < 0.0006). Expression of this antigen on IgG was also significantly different from normal in the other arthritic groups (P < 0.02), but the changes were much less than that observed for RA. In early RA, sLe^x was inversely correlated with parameters used to measure disease activity. This was not observed with the established RA, where there was weak positive association. These preliminary results indicate that a change in sLe^x expression on IgG is an early finding in the development of RA, which may be important in the development of the disease or for predicting its outcome.     

Inhibition of P-selectin-mediated cell adhesion by a sulfated derivative of sialic acid

P-selectin, a carbohydrate-binding cell adhesion molecule expressed on activated endothelial cells and platelets, plays a key role in the recruitment of leukocytes to inflammatory and hemorrhagic sites. It simultaneously recognizes a sialic acid-containing carbohydrate chain and the sulfated tyrosine residues of a specific counter-receptor expressed on the leukocyte surface. We examined the inhibitory effects of a synthetic sulfated derivative of sialic acid (NMSO3) on P-selectin-mediated cell adhesion and found the following: (1) P-selectin/IgG chimera bound to immobilized NMSO3. (2) The binding of P-selectin/IgG chimera to purified P-selectin glycoprotein ligand-1 was inhibited by soluble NMSO3. (3) The adhesion of HL60 cells to P-selectin-expressing CHO cells was inhibited by NMSO3. (4) NMSO3 inhibited P-selectin-induced tumor necrosis factor-alpha production in monocytes and activated platelet-induced generation of reactive oxygen species in neutrophils. In conclusion, NMSO3 acts as a specific inhibitor for P-selectin-mediated cell adhesion and for adhesion-dependent leukocyte activation.     

PSGL-1 derived from human neutrophils is a high-efficiency ligand for endothelium-expressed E-selectin under flow

P-selectin glycoprotein ligand-1 (PSGL-1) has been proposed as an important tethering ligand for E-selectin and is expressed at a modest level on human leukocytes. Sialyl Lewis x (sLe^x)-like glycans bind to E-selectin and are expressed at a relatively high level on circulating leukocytes. It is unclear whether PSGL-1 has unique biochemical attributes that contribute to its role as an E-selectin ligand. To probe this issue, we conjugated microspheres with either sLe^x or PSGL-1 purified from myeloid cells (neutrophils and HL-60) and compared their adhesion to endothelial expressed E-selectin under defined shear conditions. We found that both sLe^x and PSGL-1 microspheres adhere to 4 h of IL-1-activated human umbilical vein endothelial cells predominantly through E-selectin. Analysis of the adhesion revealed that the rate of initial tethering of the PSGL-1 microspheres to E-selectin was significantly greater than the rate of initial tethering of the sLe^x microspheres despite the fact that the sLe^x microspheres tested had higher ligand densities than the PSGL-1 microspheres. We also found that pretreatment of the PSGL-1 or sLe^x microspheres with HECA-452 had no significant effect on initial tethering to E-selectin. These results support the hypotheses that 1) PSGL-1 is a high-efficiency tethering ligand for E-selectin, 2) ligand biochemistry can significantly influence initial tethering to E-selectin, and 3) PSGL-1 tethering to E-selectin can occur via non-HECA-452 reactive epitopes. adhesion; leukocyte; inflammation     

L-asparaginase inhibits invasive and angiogenic activity and induces autophagy in ovarian cancer

Recent work identified L‐asparaginase (L‐ASP) as a putative therapeutic target for ovarian cancer. We suggest that L‐ASP, a dysregulator of glycosylation, would interrupt the local microenvironment, affecting the ovarian cancer cell—endothelial cell interaction and thus angiogenesis without cytotoxic effects. Ovarian cancer cell lines and human microvascular endothelial cells (HMVEC) were exposed to L‐ASP at physiologically attainable concentrations and subjected to analyses of endothelial tube formation, invasion, adhesion and the assessment of sialylated proteins involved in matrix‐associated and heterotypic cell adhesion. Marked reduction in HMVEC tube formation in vitro, HMVEC and ovarian cancer cell invasion, and heterotypic cell‐cell and cell‐matrix adhesion was observed (P < 0.05–0.0001). These effects were associated with reduced binding to ß1integrin, activation of FAK, and cell surface sialyl Lewis^X (sLe^x) expression. No reduction in HMVEC E‐selectin expression was seen consistent with the unidirectional inhibitory actions observed. L‐ASP concentrations were non‐toxic to either ovarian cancer or HMVEC lines in the time frame of the assays. However, early changes of autophagy were observed in both cell types with induction of ATG12, beclin‐1, and cleavage of LC‐3, indicating cell injury did occur. These data and the known mechanism of action of L‐ASP on glycosylation of nascent proteins suggest that L‐ASP reduces of ovarian cancer dissemination and progression through modification of its microenvironment. The reduction of ovarian cancer cell surface sLe^x inhibits interaction with HMVEC and thus HMVEC differentiation into tubes, inhibits interaction with the local matrix reducing invasive behaviour, and causes cell injury initiating autophagy in tumour and vascular cells.     

L-Selectin Ligands That Are O-glycoprotease Resistant and Distinct from MECA-79 Antigen are Sufficient for Tethering and Rolling of Lymphocytes on Human High Endothelial Venules

During the process of lymphocyte recirculation, lymphocytes bind via L-selectin to sulfated sialyl-Lewis^x (sLe^x)–containing carbohydrate ligands expressed on the surface of high endothelial venules (HEV). We have examined the expression of sLe^x on HEV using a panel of mAbs specific for sLe^x and sLe^x-related structures, and have examined the function of different sLe^x-bearing structures using an in vitro assay of lymphocyte rolling on HEV. We report that three sLe^x mAbs, 2F3, 2H5, and CSLEX-1, previously noted to bind with high affinity to glycolipid-linked sLe^x, vary in their ability to stain HEV in different lymphoid tissues and bind differentially to O-linked versus N-linked sLe^x on glycoproteins. Treatment of tissue sections with neuraminidase abolished staining with all three mAbs but slightly increased staining with MECA-79, a mAb to a sulfation-dependent HEV-associated carbohydrate determinant. Treatment of tissue sections with O-sialoglycoprotease under conditions that removed the vast majority of MECA-79 staining, only partially reduced staining with the 2F3 and 2H5 mAbs. Using a novel rolling assay in which cells bind under flow to HEV of frozen tissue sections, we demonstrate that a pool of O-sialoglycoprotease–resistant molecules is present on HEV that is sufficient for attachment and rolling of lymphocytes via L-selectin. This interaction is not inhibited by the mAb MECA-79. Furthermore, MECA-79 mAb blocks binding to untreated sections by only 30%, whereas the sLe^x mAb 2H5 blocks binding by ~60% and a combination of MECA-79 and 2H5 mAb blocks binding by 75%. We conclude that a pool of O-glycoprotease-resistant sLe^x-like L-selectin ligands exist on human HEV that is distinct from the mucin-associated moieties recognized by MECA-79 mAb. We postulate that these ligands may participate in lymphocyte binding to HEV.     

CD44v4 is a major E-selectin ligand that mediates breast cancer cell transendothelial migration

Background

Endothelial E-selectin has been shown to play a pivotal role in mediating cell–cell interactions between breast cancer cells and endothelial monolayers during tumor cell metastasis. However, the counterreceptor for E-selectin and its role in mediating breast cancer cell transendothelial migration remain unknown.

Methodology/Principal Findings

By assessing migration of various breast cancer cells across TNF-α pre-activated human umbilical vein endothelial cells (HUVECs), we found that breast cancer cells migrated across HUVEC monolayers differentially and that transmigration was E-selectin dependent. Cell surface labeling with the E-selectin extracellular domain/Fc chimera (exE-selectin/Fc) showed that the transmigration capacity of breast cancer cells was correlated to both the expression level and localization pattern of E-selectin binding protein(s) on the tumor cell surface. The exE-selectin/Fc strongly bound to metastatic MDA-MB-231, MDA-MB-435 and MDA-MB-468 cells, but not non-metastatic MCF-7 and T47D cells. Binding of exE-selectin/Fc was abolished by removal of tumor cell surface sialyl lewis x (sLe^x) moieties. Employing an exE-selectin/Fc affinity column, we further purified the counterreceptor of E-selectin from metastatic breast cancer cells. The N-terminal protein sequence and cDNA sequence identified this E-selectin ligand as a ∼170 kD human CD44 variant 4 (CD44v4). Purified CD44v4 showed a high affinity for E-selectin via sLe^x moieties and, as expected, MDA-MB-231 cell adhesion to and migration across HUVEC monolayers were significantly reduced by down-regulation of tumor cell CD44v4 via CD44v4-specific siRNA.

Conclusions/Significance

We demonstrated, for the first time, that breast cancer cell CD44v4 is a major E-selectin ligand in facilitating tumor cell migration across endothelial monolayers. This finding offers new insights into the molecular basis of E-selectin–dependent adhesive interactions that mediate breast cancer cell transendothelial metastasis.     

An improved ELISA for the determination of sialyl Lewisx structures on purified glycoconjugates

The membrane carbohydrate antigen, sialyl Lewis x (sLe^x), is involved in cellular adhesive interactions in many diseases, such as cancer, inflammation and thrombosis. This antigen is also found on soluble macromolecules, such as serum glycoproteins, but the precise role of soluble sLe^x in modifying disease processes, or reflecting the pathological changes is still unclear. Although methods were previously reported for the measurement of soluble sLe^x, many of these were not well characterised, measurements were mainly made on mixtures of molecules, and the anti-sLe^x antibodies were used at concentrations that made the assay expensive. In this study an ELISA has been devised that detects sLe^x in purified soluble glycoconjugates using the anti-sLe^x antibody, CSLEX 1. Commercially-available haptoglobin (Hp) and synthetic complexes of Lewis antigens with polyacrylamide were used as model substances in developing the procedure. Key steps were washing the antibody/antigen complex with ten times diluted salt solution to prevent dissociation of the complex and the use of bovine serum albumin for blocking non-specific interactions. The assay was shown to be very specific, its precision was in the range 6–12%, and it could detect less than a pmol of sLe^x. It could also distinguish between different densities of sLe^x on the same amount of glycoconjugate. Determination of sLe^x in Hp isolated from small groups of healthy individuals, cancer patients, and rheumatoid arthritis sufferers suggested that the antigen expression is increased in disease. This method, which is an improvement on those previously described, will be useful for determining sLe^x in many different types of soluble glycoconjugate, and used in combination with synthetic carbohydrate polyacrylamide complexes, will help to standardize measurements of soluble sLe^x in the future.     

P-selectin Cross-links PSGL-1 and Enhances Neutrophil Adhesion to Fibrinogen and ICAM-1 in a Src Kinase-Dependent,but GPCR-Independent Mechanism

Endothelial and platelet P-selectin (CD62P) and leukocyte integrin αMβ2 (CD11bCD18, Mac-1) are cell adhesion molecules essential for host defense and innate immunity. Upon inflammatory challenges, P-selectin binds to PSGL-1 (P-selectin glycoprotein ligand-1, CD162) to mediate neutrophil rolling, during which integrins become activated by extracellular stimuli for their firm adhesion in a G-protein coupled receptor (GPCR)-dependent mechanism. Here we show that cross-linking of PSGL-1 by dimeric or multimeric forms of platelet P-selectin, P-selectin receptor-globulin, anti-PSGL-1 mAb and its F(ab’)2 induced adhesion of human neutrophils to fibrinogen (Fg) and intercellular cell adhesion molecule-1 (ICAM-1, CD54) and triggered a moderate clustering of αMβ2, but monomeric forms of soluble P-selectin and anti-PSGL-1 Fab did not. Interestingly, P-selectin did not induce a detectable interleukine-8 (IL-8) secretion (&lt;0.1 ng/ml) in 30 minutes, whereas a high concentration of IL-8 (>50 ng/ml) was required to increase neutrophil adhesion to Fg. P-selectin-induced neutrophil adhesion was significantly inhibited by PP2 (a Src kinase inhibitor), but not by Pertussis toxin (PTX; a GPCR inhibitor). Activated platelets also increased neutrophil binding to fibrinogen and triggered tyrosine phosphorylation of cellular proteins. Our results indicate that P-selectin-induced integrin activation (Src kinase-dependent) is distinct from that elicited by cytokines, chemokines, chemoattractants (GPCR-dependent), suggesting that these two signal transduction pathways may cooperate for maximal activation of leukocyte integrins.