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

Glioblastoma (GBM) is the most frequent and lethal primary brain tumor, associated with a median survival of only 12-15 months after first diagnosis. The identification of new molecular targets and new therapeutic approaches are required for the development of effective treatments. Based on our previous results, a new therapeutic target for GBM treatment is potentially represented by the regulatory subunit R2A of protein kinase A (PKA). In rodent and human GBM cells this subunit presents a specific localization on Golgi apparatus, that is not detectable in the healthy brain parenchyma. Moreover, PKA R2A is significantly overexpressed in human GBM compared to non-tumor brain tissue. We previously demonstrated that siRNA-mediated R2A downregulation induces a reduction in cell viability and motility, and also fragmentation of Golgi apparatus in human GBM cells. In the present study the 3rd generation lentiviral vector cPPT.hCMV.EGFP was tested for transduction of U87 human GBM cell line and primary GBM cell cultures. Recombinant lentiviral vectors were also generated for expression of short hairpin RNA (shRNAs) targeting PKA R2A. This lentiviral system represents an efficient strategy for shRNA delivery and downregulation of PKA R2A expression. 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. 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 protein. Western blot analysis demonstrated an efficient reduction of R2A expression induced by all generated recombinant vectors, both after cell transfection and cell transduction. The 3rd generation lentiviral cPPT.hCMV.EGFP vector proved to efficiently transduce human GBM cells and human primary GBM spheres. Moreover, 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.