Abstract 1529: Knockdown of cathepsin B and uPAR inhibits CD151 and α3β1 integrin-mediated cell adhesion in glioma

Abstract

Abstract Glioma is a highly complex brain tumor characterized by deregulation of proteins and genes that play important roles in tumor metastasis. CD151, a member of the tetraspanin family of proteins, tightly associates with integrins and accelerates cell adhesion as a modulator of actin cytoskeletal reorganization. Cathepsin B and uPAR are both overexpressed in gliomas and postulated to play central roles in the mediation of glioma metastasis. In the present study, cathepsin B and uPAR were downregulated in U251 and 4910 glioma cells using siRNA plasmid constructs. We evaluated the involvement of CD151 and its associated signaling molecules in adhesion potential. Immunoblot analysis revealed efficient downregulation of cathepsin B and uPAR after siRNA treatments. In addition, siRNA treatments significantly inhibited glioma cell adhesion to laminin as compared to other extracellular matrix proteins. Brain glioma tissue array analysis revealed the expression of CD151 in several tumor samples when compared with normal brain tissue. Further, treatment of glioma cells with cathepsin B and uPAR siRNA led to the downregulation of CD151 and laminin-binding integrins, α3 and β1. Co-localization and immunoprecipitation experiments revealed that downregulation of cathepsin B and uPAR decreased the interaction of CD151 with uPAR/cathepsin B. Transcriptional suppression of CD151 and α3 and β1 integrins using siRNA significantly decreased glioma cell adhesion to laminin. In addition, downregulation of cathepsin B, uPAR, CD151, α3 and β1 significantly decreased the expression of N-cadherin and β-catenin. Studies on the downstream signaling cascade of uPAR/CD151 have shown that phosphorylation of SRC, FAK and Paxillin were reduced with knockdown of cathepsin B, uPAR and CD151. Treatment with the bicistronic construct reduced CD151, α3β1 integrin, N-cadherin and β-catenin expression levels in pre-established glioma tumors in nude mice as well as in tissue lysates. In conclusion, our results show that downregulation of cathespin B and uPAR alone and in combination inhibit glioma cell adhesion by downregulating CD151 and its associated signaling molecules in vitro and in vivo. This study may be useful in dissecting the pathways involved in tumor cell adhesion and for the identification of novel target proteins for therapeutic intervention of gliomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1529. doi:10.1158/1538-7445.AM2011-1529