Abstract 1080: Targeting the PI3K-Akt and NF-κB pathways as a combination therapy in blocking prostate cancer progression

Abstract

Akt/protein kinase B and transcription factor NF-κB are important signaling molecules and key survival factors involved in the control of cell proliferation, apoptosis and oncogenesis. Although Akt and NF-κB are components of separate signaling pathways, Akt can induce cell survival signal through NF-κB activation. Previous studies from our laboratory have shown constitutive activation of NF-κB/p65 and PI3K-Akt in clinical prostate cancer specimens and in autochthonous mouse model of prostate cancer. In this study we investigated if these two signaling pathways converge and whether dual targeting is effective in blocking disease progression. Using tissue samples obtained during transurethral prostatic resection and paraffin-embedded sections of benign and malignant prostate tissue, we first analyzed nuclear levels of NF-κB/p65, native/activated Akt levels, and evaluated co-localization of these two proteins. Compared to benign tissue, cancer specimens exhibited constitutive Akt and NF-κB/p65 activation which was more pronounced in high-grade cancer (Gleason grade 7-10). Immunohistochemical analyses further confirmed a progressive increase in the activated form of Akt and NF-κB/p65 in the nucleus of cancer tissues compared to benign specimens, and exhibited co-localization of these proteins in a subset of aggressive cancer. Utilizing androgen-responsive prostate cancer LNCaP cells, with increased Akt activity, and androgen-refractory PC-3 cells harboring high levels of Akt and NF-κB. Individual treatment of cells with Akt Inhibitor VIII (0.075-1.8µM) and NF-κB inhibitor Parthenolide (0.32-60µM) for 24-72 h demonstrated partial suppressive effect in cell growth. Strikingly, concurrent blocking of Akt and NF-κB/p65 at 1:10 molar ratio resulted in potentiated toxicity with marked inhibition in proliferation and induction of cell cycle arrest. Interruption of Akt activation using dominant-negative approach resulted in significant inhibition in Akt-phosphorylation (Ser473) and induction of apoptosis in LNCaP cells, whereas ectopic expression of NF-κB/p65 increased doubling time and invasiveness in these cells, which was abrogated by combinational treatment. Furthermore, combination treatment decreased the expression of p-IKK (Ser176/180), p-NF-κB/p65 (Ser536), and p-Akt (Ser473) and downstream targets viz. cyclin D1, Bcl-2, VEGF and MMP9 in both cell lines. In vivo administration of Inhibitor VIII and Parthenolide at 1:10 ratio to PC-3 tumor xenograft for 8 weeks resulted in marked decrease in tumor volume and reduced expression of target genes, compared to individual treatments. These results suggest that nuclear co-localization of Akt and NF-κB/p65 may be developed as prognostic biomarker and concurrent targeting of these molecules by combination of pharmacological inhibitors is an effective approach and is more efficacious than use of single agent blocking prostate cancer progression. Citation Format: Eswar Shankar, Rajnee Kanwal, Aditi Goel, Xiaoping Yang, Sanjeev Shukla, Gregory T. MacLennan, Pingfu Fu, Anant Madabhushi, Parameswaran Ramakrishnan, Sanjay Gupta. Targeting the PI3K-Akt and NF-κB pathways as a combination therapy in blocking prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1080. doi:10.1158/1538-7445.AM2017-1080