Epidermal growth factor (EGF) receptor (EGFR) signalling is implicated in tumour invasion and metastasis. However, whether there are EGFR signalling pathways specifically used for tumour invasion still remains elusive. Overexpression of Arf6 and its effector, AMAP1, correlates with and is crucial for the invasive phenotypes of different breast cancer cells. Here we identify the mechanism by which Arf6 is activated to induce tumour invasion. We found that GEP100/BRAG2, a guanine nucleotide exchanging factor (GEF) for Arf6, is responsible for the invasive activity of MDA-MB-231 breast cancer cells, whereas the other ArfGEFs are not. GEP100, through its pleckstrin homology domain, bound directly to Tyr1068/1086-phosphorylated EGFR to activate Arf6. Overexpression of GEP100, together with Arf6, caused non-invasive MCF7 cells to become invasive, which was dependent on EGF stimulation. Moreover, GEP100 knockdown blocked tumour metastasis. GEP100 was expressed in 70% of primary breast ductal carcinomas, and was preferentially co-expressed with EGFR in the malignant cases. Our results indicate that GEP100 links EGFR signalling to Arf6 activation to induce invasive activities of some breast cancer cells, and hence may contribute to their metastasis and malignancy.

Here we show that a large chondroitin sulfate proteoglycan, versican, derived from a renal adenocarcinoma cell line ACHN, binds L-selectin, P-selectin, and CD44. The binding was mediated by the interaction of the chondroitin sulfate (CS) chain of versican with the carbohydrate-binding domain of L-and P-selectin and CD44. The binding of versican to L-and P-selectin was inhibited by CS B, CS E, and heparan sulfate (HS) but not by any other glycosaminoglycans tested. On the other hand, the binding to CD44 was inhibited by hyaluronic acid, chondroitin (CH), CS A, CS B, CS C, CS D, and CS E but not by HS or keratan sulfate. A crossblocking study indicated that L-and P-selectin recognize close or overlapping sites on versican, whereas CD44 recognizes separate sites. We also show that soluble L-and P-selectin directly bind to immobilized CS B, CS E, and HS and that soluble CD44 directly binds to immobilized hyaluronic acid, CH, and all the CS chains examined. Consistent with these results, structural analysis showed that versican is modified with at least CS B and CS C. Thus, proteoglycans sufficiently modified with the appropriate glycosaminoglycans should be able to bind L-selectin, P-selectin, and/or CD44.

We previously reported that versican, a large chondroitin/dermatan sulfate (CS/DS) proteoglycan, interacts through its CS/DS chains with adhesion molecules L-and P-selectin and CD44, as well as chemokines. Here, we have characterized these interactions further. Using a metabolic inhibitor of sulfation, sodium chlorate, we show that the interactions of the CS/DS chains of versican with L-and P-selectin and chemokines are sulfationdependent but the interaction with CD44 is sulfationindependent. Consistently, versican's binding to L-and P-selectin and chemokines is specifically inhibited by oversulfated CS/DS chains containing GlcA␤1-3Gal-NAc(4,6-O-disulfate) or IdoA␣1-3GalNAc(4,6-O-disulfate), but its binding to CD44 is inhibited by all the CS/DS chains, including low-sulfated and unsulfated ones. Affinity and kinetic analyses using surface plasmon resonance revealed that the oversulfated CS/DS chains containing GlcA␤1/IdoA␣1-3GalNAc(4,6-O-disulfate) bind directly to selectins and chemokines with high affinity (K d 21.1 to 293 nM). In addition, a tetrasaccharide fragment of repeating GlcA␤1-3GalNAc(4,6-Odisulfate) units directly interacts with L-and P-selectin and chemokines and oversulfated CS/DS chains containing GlcA␤1/IdoA␣1-3GalNAc(4,6-O-disulfate) inhibit chemokine-induced Ca 2؉ mobilization. Taken together, our results show that oversulfated CS/DS chains containing GlcA␤1/IdoA␣1-3GalNAc(4,6-O-disulfate) are recognized by L-and P-selectin and chemokines, and imply that these chains are important in selectin-and/or chemokine-mediated cellular responses.Proteoglycans are ubiquitous components of cell surface membranes, basement membranes, and extracellular matrices in various tissues. They belong to a family of macromolecules that consist of core proteins to which glycosaminoglycans (GAGs), 1 sulfated polysaccharides, are attached. GAGs are linear polysaccharides made up of disaccharide units composed of hexosamine and hexuronic acid (or hexose). They are classified into chondroitin sulfate (CS), dermatan sulfate (DS), heparin, heparan sulfate (HS), keratan sulfate (KS), and hyaluronic acid (HA). Because of the high sulfate and carboxyl group content of their GAG moieties, proteoglycans have strong negative charges. This property allows them to interact with a wide range of proteins, including growth factors, enzymes, cytokines, chemokines, lipoproteins, and adhesion molecules (1, 2). We previously showed that a large CS/DS proteoglycan, versican (also called PG-M), that was derived from a renal adenocarcinoma cell line, ACHN, interacts through its CS/DS chains with adhesion molecules such as L-and P-selectin and CD44 (3, 4), and various chemokines (5), all of which have been implicated in leukocyte trafficking. Versican possesses a hyaluronic acid-binding domain at its N terminus, a GAG attachment domain in the middle, and a set of epidermal growth factor-like, C-type lectin-like, and complement regulatory protein-like domains at its C terminus (6). Alternative splicing of the versican gene generates four ver...

mammalian sphingolipids is sphingosine ( trans -4-sphingenine, d18:1 4t ). Smaller amounts of other sphingoid bases, such as sphinganine (dihydrosphingosine, d18:0) and phytosphingosine (4-hydroxysphinganine, t18:0), are encountered frequently. In higher plants, the structures of sphingoid bases are more complicated than in mammals, because they can be desaturated at the C8-position by a ⌬ 8-sphingolipid desaturase, yielding cis -and trans -isomers of ⌬ 8-unsaturated sphingoid bases (d18:2 4t,8c(t) ) ( 3, 4 ). 9-Methyl-trans -4, trans -8-sphingadienine (d19:2 4t,8t ) is a typical structure found in yeasts ( 5 ). Sphingolipids of marine invertebrates have unique triene types of sphingoid bases with a conjugated diene such as 2-amino-4,8,10-octatriene-1, 3-diol (d18:3 4,8,10 ) and 2-amino-9-methyl-4,8,10-octatriene-1, 3-diol (d19:3 4,8,10 ) ( 6 ). Therefore, sphingolipids having various structures of sphingoid bases are ingested daily from foodstuffs ( 7-9 ).Dietary sphingolipids have gained attention for their potential to protect against infl ammation and cancers in the gut ( 10-13 ). One plausible mechanism for these effects may be via the hydrolysis of complex sphingolipids to bioactive ceramides and sphingosine, because those breakdown products are known to play important roles as intracellular mediators ( 14, 15 ). We previously demonstrated that dietary maize and yeast sphingolipids with sphingoid bases distinct from those of mammals are able to prevent the formation of aberrant crypt foci in 1,2-dimethylhydrazine-treated mice ( 16,17 ). We further showed that sphingoid bases prepared from various dietary sources can induce apoptosis in cancer cells (18)(19)(20).In early studies, Nilsson ( 21-23 ) investigated the digestion and intestinal absorption of sphingolipids containing sphingosine and sphinganine. Dietary sphingolipids can be hydrolyzed to their components, such as sphingoid bases, fatty acids, and the polar head group, by intestinal enzymes and are then taken up by mucosal cells ( 24,25 ). A large portion of sphingosine absorbed Sphingolipids are ubiquitous in all eukaryotic organisms and constitute a family of compounds that have a sphingoid base (long-chain base) with an amide-linked fatty acid and a polar head group. It is well known that there are diverse structures of sphingoid bases in nature ( Fig. 1 ) ( 1, 2 ). The most common sphingoid base of