A Synthetic Polyvalent Ligand for α5β1 Integrin Activates Components of the Urothelial Carcinoma Cell Response to Bacillus Calmette-Guérin

Abstract

Prior study has shown that bacillus Calmette-Guérin binds to and cross-links α5β1 integrins present on the surface of urothelial carcinoma cells. Antibody mediated cross-linking of α5β1 integrins can reproduce signal transduction, gene transactivation and phenotypic changes, similar to those observed in response to bacillus Calmette-Guérin. We evaluated the effect of a synthetic polyvalent ligand for α5β1 on these elements of the tumor response to bacillus Calmette-Guérin. The consensus α5β1 integrin binding tripeptide RGD was linked to a MAP8 backbone to result in an octavalent construct targeting α5β1 integrin. RGD-MAP8 was used to determine its effect on signaling pathway activation (nuclear factor-κB, NRF2 and CEBP), gene expression (p21, interleukin-6 and 8, CXCL1, CXCL2 and CCL20) and cytotoxicity (trypan blue exclusion and HMGB1 release) in human urothelial carcinoma cells. Results were compared to those of treatment with bacillus Calmette-Guérin or the missense peptide GRD-MAP8. The RDG-MAP8 construct significantly increased nuclear factor-κB signaling and p21 expression relative to controls. Compared to bacillus Calmette-Guérin treatment, only p21 expression was comparable for cells treated with RGD-MAP8, averaging 70% of bacillus Calmette-Guérin induced expression. RGD-MAP8 failed to have a significant effect on CEBP or NRF2 activation, gene expression or cell viability. Intracellular signaling, gene transactivation and phenotypic changes in response to RGD-MAP8 were qualitatively and quantitatively different than those observed in response to bacillus Calmette-Guérin. Results suggest that while α5β1 integrin cross-linking contributes to the bacillus Calmette-Guérin response, it alone is insufficient to duplicate the full spectrum of bacillus Calmette-Guérin induced changes in urothelial carcinoma cell biology.