881 A SYNTHETIC POLYVALENT LIGAND FOR α5β1 INTEGRIN ACTIVATES COMPONENTS OF THE UROLTHELIAL CARCINOMA CELL RESPONSE TO BCG

Abstract

INTRODUCTION AND OBJECTIVES: Prior work has demonstrated that BCG binds to, and crosslinks, 5 1 integrins present on the surface of urothelial carcinoma cells (UCC). Antibody mediated crosslinking of 5 1 integrins can reproduce signal transduction, gene transactivation, and phenotypic changes similar to those observed in response to BCG. The purpose of the present study was to evaluate the effect of a synthetic polyvalent ligand for 5 1 on these elements of the tumor response to BCG. METHODS: The consensus 5 1 integrin binding tri-peptide “RGD” was linked to a MAP8 backbone to result in an octavalent construct targeting 5 1 integrin. Purified RGD-MAP8 was then used in dose response experiments to determine its effect on signaling pathway activation (NFKB, NF2, and CEBP), gene expression (p21, IL6, IL8, CXCL1, CXCL2, CCL20), and phenotypic changes (trypan blue exclusion, HMGB1 release) in UCC associated with BCG treatment. The effect of the construct was tested in the human UCC lines 253J and T24. The scrambled sequence GRD-MAP8 served as a negative control with BCG exposure serving as a positive control. RESULTS: The RGD-MAP8 construct activated NFKB, NF2, and CEBP signaling pathways in the 2 cell lines, in a sequence specific, dose-response manner, to maximum average values of 89%, 60% and 42% of BCG exposed positive controls respectively. p21 expression in response to RDG-MAP8 averaged 70% of positive controls. Although the synthetic construct transactivated cytokine gene expression in 2/5 and 5/5 target genes in the 253J and T24 cell line respectively, expression levels averaged only 18% of BCG treated control values. RGD-MAP8 exposure resulted in loss of trypan blue membrane integrity with peak values of 65% of BCG controls. In contrast to BCG exposure, RGD-MAP8 treatment did not result in HMGB1 release. None of the endpoints demonstrated a response to the scrambled sequence control. CONCLUSIONS: Treatment of UCC with a synthetic, polyvalent compound designed to crosslink cell surface 5 1 integrin activates intracellular signaling pathways and transactivates the expression of some BCG responsive genes. However, the magnitude of both signal induction and gene transactivation was less than observed in response to BCG. Moreover, some genes appeared unresponsive to RGD-MAP8 and no HMGB1 release (necrosis marker) was observed. These results suggest that while 5 1 integrin crosslinking contributes to the BCG response, it alone is insufficient to duplicate the full spectrum of BCG induced changes in UCC biology.