Anti-tumor antibody BR96 blocks cell migration and binds to a lysosomal membrane glycoprotein on cell surface microspikes and ruffled membranes.

Abstract

Abstract. BR 96 is an internalizing antibody that binds to Lewis Y (LeY), a carbohydrate determinant expressed at high levels on many human carcinomas (Hellstr6m, I., H. J. Garrigues, U. Garfigues, and K. E. Hellstr6m. 1990. Cancer Res. 50:2183-2190. Breast carcinoma cell lines grown to confluence bind less BR96 than subconfluent cultures (Garrigues, J., U. Garrigues, I. Hellstr6m, and K. E. Hellstr6m. 1993. Am. J. Path. 142:607-622). However, when the confluent cells are induced to migrate by scratch wounding, they again bind BR96 suggesting that antigens bearing the Ley determinant may promote cell migration. In the present study, BR96 was found to be highly enriched on microspikes and ruffled membranes, cell surface structures involved in cell migration. In addition, BR96 was a potent inhibitor of cell migration in vitro. When stationary BR96 treated cells were exposed to fresh culture media, membrane ruffles and microspikes developed at the cell margin and migration resumed. Immunogold microscopy showed that BR96 antigens were enriched on these membrane protrusions.BR96 cell surface immunoprecipitation analysis of 3H-glucosamine labeled breast carcinoma cells identified antigens with approximate molecular weights of 135 kd (upper antigen) and 85 kd (lower antigen). A short amino terminal sequence (8 residues) of the upper antigen matched that of human lysosomal membrane glycoprotein 1 (LAMP-l). In addition, the upper antigen was detected on immunoblots probed with anti-LAMP-l, and within the intracellular compartment BR96 was found predominantly in endosomes and lysosomes.A soluble LAMP-1/immunoglobulin fusion protein (LAMP-1/Ig) was transiently expressed in both BR96 binding and nonbinding cell lines. Immunoblot analysis of LAMP-1/Ig's from the various cell lines showed that (a) acquisition of the BR96 epitope is probably controlled at the level of polylactosamine modification (e.g., fucosylation) rather than LAMP-1 gene expression; (b) alternate forms of LAMP-1/Ig comigrate with the lower BR96 antigen raising the possibility that it may be a degradation product of the upper antigen; and (c) LAMP-1/Ig expressed in 3396 breast carcinoma cells has approximately 30-fold more BR96 epitopes than LAMP-1/Ig from non-tumorigenic mammary epithelial cells. Together these data indicate that a major BR96 antigen, LAMP-l, is present on unique cell surface domains involved in cell locomotion as well as membranes of the endocytic compartment. Altered glycosylation of LAMP-1 expressed in transformed cells may contribute to their ability to disseminate. MANY ligands have been identified that can regulate cell locomotion (Turley, 1992). Some studies indicate that altered cell migration is associated with aberrant glycosylation. In a number of instances, highly metastatic tumor cells were found to express more polylactosamine oligosaccharides (Gal/31-4GlcNAc/31-3), than normal or poorly metastatic ones (Hubbard, 1987;Pierce and Arango, 1986;Yamashita et al., 1984;Yousefi et al., 1991). Poly N-acetyllactosamine chains are oft...

Carbohydrate Mimicking Peptides as Inhibitors of Angiogenesis and Metastasis

“…Antibodies BR55-2 and BR15-6A directed to LeY significantly decreased the adhesion in a dose-dependent fashion, implicating the involvement of LeY in adhesion of these breast carcinoma cells. These data indicate that different human adenocarcinoma cells interact with EC via E-selectin-mediated and E-selectin-independent pathways in agreement with previously published reports that some invasive breast carcinoma cells do not interact with TNF-c(x-stimulated EC under dynamic conditions, but adhere to resting and stimulated EC via an E-selectin independent mode following static incubation (12,13).…”

Hypoxic Activation of the PERK/eIF2α Arm of the Unfolded Protein Response Promotes Metastasis through Induction of LAMP3

“…However, the biologic function of LAMP3 in migration and metastasis in tumor cells is not understood. LAMP1 was shown to be required for proper formation of membrane ruffles and filopodia in migrating tumor cells (45) and it is possible that LAMP3 exerts a similar function in response to hypoxia. A role for LAMP3 in cell-cell adhesion has also been proposed as it is a highly glycosylated protein that is often localized to the cell surface of tumor cells (42,46), and other LAMP family members have already been implicated in cell adhesion (47).…”

Serological and immunochemical analysis of Lewis Y (Ley) blood group antigen expression in epithelial ovarian cancer

“…As Garrigues et al (1994) had demonstrated that the lysosomal glycoprotein LAMP-1 was the major Ley-bearing component in 3396 breast cancer cells, we examined the expression of LAMP-1 in ovarian cancer cell lines and its possible relationship to the faster-migrating Ley-reactive species. By radioimmunoprecipitation analysis, OVCAR-3 cells were found to be LAMP-1 negative (Fig.…”