Abstract 2312: Targeting beta1 integrin potentiates antiangiogenic therapy and inhibits growth of bevacizumab-resistant glioblastoma multiforme.
Authors: Carbonell W. Shawn , Lay Michael De , Jahangiri Arman , Park Catherine C. , Aghi Manish 
Published: August 2014

INTRODUCTION: Antiangiogenic therapies like bevacizumab hold promise for cancer treatment. However, the development of acquired resistance, including an aggressive mesenchymal phenotype, limit utility of these agents. We previously published a report in which microarray and PCR analysis revealed beta1 integrin to be upregulated in bevacizumab-resistant glioblastomas (BRGs). Because the beta1 integrin subunit mediates tumor-microenvironment interactions, we further investigated its role in mesenchymal-type resistance to anti-angiogenic therapy.

METHODS: Immunostaining of BRG specimens and genetic or pharmacologic beta1 targeting in BRG cells and BRG-derived xenografts were used to study the role of beta1 integrin in anti-angiogenic therapy resistance in vitro and in vivo. Cells were transduced to express beta1-GFP fusion protein to measure beta1 integrin turnover in focal adhesions using fluorescence recovery after photobleaching (FRAP).

RESULTS: Beta1 integrin and downstream beta1-effector focal adhesion kinase immunostaining were upregulated in 75% and 86% of BRGs, resepectively, compared to pre-treatment paired specimens. Flow cytometry revealed 8-fold upregulation of beta1 integrin in BRG-derived primary GBM cells compared to cells from bevacizumab-naïve GBMs (P&lt;0.05). FRAP revealed more rapid beta1 integrin turnover in focal adhesion kinases in BRG-derived cells than in bevacizumab-naïve GBM cells, with 4-fold more time to achieve 37% recovery in bevacizumab-naïve GBM cells than in BRG-derived cells expressing a beta1-GFP fusion protein (95 versus 398 seconds; P&lt;0.05).

Hypoxia, which was increased 80% (as evidenced by CA9 immunostaining) (P&lt;0.05) after bevacizumab resistance, increased beta1 integrin expression 30% in cultured bevacizumab-naïve GBM cells (P&lt;0.01). Two hours of incubation with 10 and 100 ng/mL VEGF reduced adhesion of GBM cells to beta1 ligands fibronectin, collagen IV, and laminin by 10-50% in a dose-dependent fashion (P&lt;0.05).

BRGs demonstrated aggressive mesenchymal-like phenotype in vitro and growth of subcutaneous BRG xenografts was attenuated by beta1 antibody OS2966 (P&lt;0.05). Addition of OS2966 allowed 20-fold dose reduction of bevacizumab per cycle in subcutaneous GBM-derived xenografts (P&lt;0.05). Intracranial delivery of OS2966 through osmotic pumps in mice carrying intracranial BRG-derived xenografts over 28 days increased tumor cell apoptosis, decreased tumor cell invasiveness, and altered tumor cell morphology (P&lt;0.05).

CONCLUSIONS: Beta1 integrin expression and turnover in focal adhesion kinases is upregulated in BRGs due to 2 possible mechanisms - hypoxic upregulation and bevacizumab-induced VEGF depletion eliminating VEGF-mediated inhibition of beta1 activity. Beta1 inhibition is well tolerated in vivo and holds promise for disrupting anti-angiogenic therapy resistance.

Citation Format: W. Shawn Carbonell, Michael De Lay, Arman Jahangiri, Catherine C. Park, Manish Aghi. Targeting beta1 integrin potentiates antiangiogenic therapy and inhibits growth of bevacizumab-resistant glioblastoma multiforme. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2312. doi:10.1158/1538-7445.AM2013-2312