Inhibition of p21-activated Kinase 6 (PAK6) Increases Radiosensitivity of Prostate Cancer Cells

Abstract

For patients with high-risk localized prostate cancer, biochemical and clinical relapse after radiotherapy remains a significant problem. New therapeutic target for radiation sensitization is needed. p21-activated kinase 6 (PAK6), a serine/threonine kinase belonging to the p21-activated kinase (PAK) family, was first identified as an androgen receptor (AR)-interacting protein able to inhibit AR-mediated transcriptional responses. In this study, we investigated the role of PAK6 in radiation-induced cell death in human PC-3 and DU-145 prostate cancer cells. We used a short hairpin RNA (shRNA) strategy to stably knock down PAK6 in PC-3 and DU-145 cells. Radiation sensitivities were compared in PAK6 stably knockdown cells and in scrambled shRNA-expressing cells. 6 Gy of irradiation upregulated PAK6 mRNA and protein levels in PC-3 and DU-145 cells. Transfection with PAK6 shRNA resulted in a significant decrease of PAK6 mRNA and protein expression. After irradiation, an increased percentage of apoptotic cells and an increased cleaved caspase 3 level in parallel with a decreased cell viability and a reduced clonogenic survival was shown. Furthermore, transfection with PAK6 shRNA blocked cells in a more radiosensitive G2/M phase and increased levels of DNA double-strand breaks as indicated by a higher amount of γ-H2AX in irradiated cells. We further explored the potential mechanisms by which PAK6 mediates resistance to radiation-induced apoptosis. Inhibition of PAK6 caused a decrease in Ser112 phosphorylation of BAD, a proapoptotic member of the Bcl-2 family, which led to enhanced binding of BAD to Bcl-2 and Bcl-XL and release of cytochrome c culminating into caspase activation and cell apoptosis. The combination of PAK6 inhibition and irradiation resulted in significantly decreased survival of prostate cancer cells. The underlying mechanisms by which targeting Pak6 may improve radiation response seem to be multifaceted, and involve alterations in cell cycle distribution and impaired DNA double-strand break repair as well as relieved BAD phosphorylation. Additional studies are ongoing to validate PAK6 as a target for radiation sensitization in animal models of prostate cancer.