Loss of phosphodiesterase 4D mediates acquired triapine resistance via Epac-Rap1-Integrin signaling

// Walter Miklos 1 , Petra Heffeter 1, 3 , Christine Pirker 1 , Sonja Hager 1 , Christian R. Kowol 2, 3 , Sushilla van Schoonhoven 1 , Mirjana Stojanovic 1 , Bernhard K. Keppler 2, 3 , Walter Berger 1, 3 1 Department of Medicine I, Institute of Cancer Research and Comprehensive Cancer Center, Medical University of Vienna, A-1090 Vienna, Austria 2 Institute of Inorganic Chemistry, University of Vienna, A-1090 Vienna, Austria 3 Research Platform “Translational Cancer Therapy Research”, University Vienna and Medical University Vienna, Vienna, Austria Correspondence to: Walter Berger, email: walter.berger@meduniwien.ac.at Keywords: triapine-resistance, phosphodiesterase, Epac, Rap1, integrin Received: March 14, 2016      Accepted: August 24, 2016      Published: September 02, 2016 ABSTRACT Triapine, an anticancer thiosemicarbazone, is currently under clinical investigation. Whereas promising results were obtained in hematological diseases, trials in solid tumors widely failed. To understand mechanisms causing triapine insensitivity, we have analysed genomic alterations in a triapine-resistant SW480 subline (SW480/tria). Only one distinct genomic loss was observed specifically in SW480/tria cells affecting the phosphodiesterase 4D ( PDE4D ) gene locus. Accordingly, pharmacological inhibition of PDE4D resulted in significant triapine resistance in SW480 cells. Hence, we concluded that enhanced cyclic AMP levels might confer protection against triapine. Indeed, hyperactivation of both major downstream pathways, namely the protein kinase A (PKA)-cAMP response element-binding protein (Creb) and the exchange protein activated by cAMP (Epac)-Ras-related protein 1 (Rap1) signaling axes, was observed in SW480/tria cells. Unexpectedly, inhibition of PKA did not re-sensitize SW480/tria cells against triapine. In contrast, Epac activation resulted in distinct triapine resistance in SW480 cells. Conversely, knock-down of Epac expression and pharmacological inhibition of Rap1 re-sensitized SW480/tria cells against triapine. Rap1 is a well-known regulator of integrins. Accordingly, SW480/tria cells displayed enhanced plasma membrane expression of several integrin subunits, enhanced adhesion especially to RGD-containing matrix components, and bolstered activation/expression of the integrin downstream effectors Src and RhoA/Rac. Accordingly, integrin and Src inhibition resulted in potent triapine re-sensitization especially of SW480/tria cells. In summary, we describe for the first time integrin activation based on cAMP-Epac-Rap1 signaling as acquired drug resistance mechanism. combinations of triapine with inhibitors of several steps in this resistance cascade might be feasible strategies to overcome triapine insensitivity of solid tumors.