Abstract A18: The integrin αvβ6 regulates PDAC cell growth and stromal cell behavior

Abstract

Abstract Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the UK. Five-year survival rates are less than 3% and haven’t improved over the last 40 years. Thus novel approaches are required urgently. We have found that over 90% of PDAC express αvβ6 and high expression of the integrin β6 gene (ITGB6) correlates with a significant reduction (Hazard Ratio 2.07; Logrank p=3.17x10-8) in overall survival from PDAC. Thus we have examined the biological functions mediated by αvβ6 in PDAC cell lines. A panel of human PDAC cell lines was incubated with αvβ6-blocking antibodies in order to define the role of αvβ6 in vitro. Some cell lines were driven into G2/M cell cycle arrest (Panc0403), whilst others died through apoptosis (CfPac1). Moreover, adhesion, migration and invasion through Matrigel were suppressed by blockade of αvβ6. Treatment of subcutaneous tumors composed of human αvβ6-positive cell lines in combination with human pancreatic stellate cells, with an αvβ6-blocking antibody resulted in significantly reduced tumor volume and growth rate in vivo, via modulation of the stromal compartment. In combination, these data suggested that targeting αvβ6 may be an effective therapeutic strategy in PDAC. To investigate the biological roles and molecular bases of αvβ6 in PDAC, cell lines were generated from PDAC tumors of a novel PDAC transgenic mouse. Two primary cell populations from PDAC tumors, abbreviated as KVC1 and KPCD3, demonstrated both epithelial and mesenchymal characteristics. Thus, cells were subsequently sorted by FACS to obtain high-αvβ6 expressing monoclonal cell lines. Resultant monoclonal cell lines expressed higher levels of αvβ6 than the parental line and expressed only epithelial markers. Transduction with lentivirus particles containing shRNA to itgb6 under a tetracycline-inducible promoter (SMARTvector, Dharmacon) was optimized for four high-αvβ6 expressing monoclonal cell lines, and doxycycline-induced knock-down of β6 protein was 70-80%, confirmed using FACS and western blotting. In vitro functional assays demonstrated that knock-down of itgb6 reduced invasion of Matrigel. To assess the role of αvβ6 in an assay that better reflected the PDAC environment, we developed 3D organotypic assays with inducible knockout PDAC combined with mouse embryo fibroblasts or mouse pancreatic stellate cells. Doxycycline treatment reduced PDAC tumor cell growth and invasion, but also reduced the contraction of the collagen-Matrigel gel matrix. Since αvβ6 activates TGFβ, and as TGFβ promotes fibroblast and stellate cell contractile propensity, current studies will determine whether this is the mechanism of how αvβ6 regulates stromal cell behavior. Citation Format: Claire S. Reader, Jennifer P. Morton, Owen J. Sansom, John F. Marshall.{Authors}. The integrin αvβ6 regulates PDAC cell growth and stromal cell behavior. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A18.