P08.69 A lentiviral-based system for targeting the regulatory subunit R2A of protein kinase A in human glioblastoma

Abstract

Introduction: Glioblastoma (GBM) is the most frequent and lethal primary brain tumor, associated with a median survival of 12-15 months after first diagnosis. The identification of new therapeutic approaches is required for the development of effective treatments. The cAMP-activated protein kinase (PKA) pathway is dysregulated in many pathological conditions, including cancer, and could potentially provide new therapeutic targets for GBM treatment. In rodent and human GBM cells the regulatory subunit R2A of PKA presents a specific localization on Golgi apparatus, that is not detectable in the healthy brain parenchyma. Moreover, downregulation of PKA R2A expression induces a reduction of GBM cell viability. In order to deeply investigate the effects of stable PKA R2A downregulation, a lentiviral-based system was developed for delivery of short hairpin RNA (shRNA) targeting PKA R2A gene to human GBM cells. The 3rd generation lentiviral vector cPPT.hCMV.EGFP was first tested for transduction of U87 human GBM cell line and primary GBM cell cultures. Then, recombinant lentiviral vectors were generated for expression of shRNA targeting PKA R2A gene. This lentiviral system represents an efficient strategy for shRNA delivery and downregulation of PKA R2A expression. Material and method: U87 human GBM cell line and primary GBM cell cultures were grown respectively as adherent cells in serum-supplemented medium and as spheres in serum-free medium. 293T cells were used for production and titration of lentiviral vectors. EGFP expression was monitored at epifluorescence microscope and western blot analysis was performed to evaluate R2A expression. Results and discussion: The lentiviral cPPT.hCMV.EGFP vector is able to efficiently transduce U87 cells and primary GBM spheres. Transduced U87 cells maintain the expression of the EGFP reporter gene for more than two weeks and no apparent influence on cell growth was observed. Expression of EGFP is detected from most, but not all, cells forming the transduced primary GBM spheres. Moreover, transduced primary GBM cells are able to form spheres and maintain the expression of the reporter gene after dissociation. Recombinant lentiviral cPPT.hCMV.EGFP-based vectors were generated for expression of shRNA.Temp1 or shRNA.Temp2, targeting different exons of PKA R2A gene, and for their co-expression with EGFP. Western blot analysis demonstrated an efficient reduction of R2A expression in cells transfected with the cis-elements of recombinant vectors and in cells transduced with the recombinant lentiviral particles. Conclusion: The 3rd generation lentiviral cPPT.hCMV.EGFP vector proved to efficiently transduce human GBM cells and primary GBM spheres. This lentiviral system allows efficient delivery of shRNA for downregulation of PKA R2A expression, thus representing a new strategy for targeting protein kinase A in human GBM cells.