Oncolytic Herpes Simplex Virus and PI3K Inhibitor BKM120 Synergize to Promote Killing of Prostate Cancer Stem-like Cells

Abstract

Novel therapies to override chemo-radiation resistance in prostate cancer (PCa) are needed. Prostate cancer sphere-forming cells (PCSCs) (also termed prostate cancer stem-like cells) likely participate in tumor progression and recurrence, and they are important therapeutic targets. We established PCSC-enriched spheres by culturing human (DU145) and murine (TRAMP-C2) PCa cells in growth factor-defined serum-free medium, and we characterized stem-like properties of clonogenicity and tumorigenicity. The efficacy of two different oncolytic herpes simplex viruses (oHSVs) (G47Δ and MG18L) in PCSCs was tested alone and in combination with radiation; chemotherapy; and inhibitors of phosphoinositide 3-kinase (PI3K), Wnt, and NOTCH in vitro ; and, G47Δ was tested with the PI3K inhibitor BKM120 in a PCSC-derived tumor model in vivo . PCSCs were more tumorigenic than serum-cultured parental cells. Human and murine PCSCs were sensitive to oHSV and BKM120 killing in vitro , while the combination was synergistic. oHSV combined with radiation, docetaxel, Wnt, or NOTCH inhibitors was not. In athymic mice bearing DU145 PCSC-derived tumors, the combination of intra-tumoral G47Δ and systemic BKM120 induced complete regression of tumors in 2 of 7 animals, and it exhibited superior anti-tumor activity compared to either monotherapy alone, with no detectable toxicity. oHSV synergizes with BKM120 in killing PCSCs in vitro , and the combination markedly inhibits tumor growth, even inducing regression in vivo .

Inhibition of Notch pathway enhances the anti-tumor effect of docetaxel in prostate cancer stem-like cells

“…PCSCs were cultured in EF20 medium composed of neurobasal medium (Invitrogen) supplemented with 3 mM L-glutamine (Mediatech), 20 ng/mL human EGF (R&D Systems), 20 ng/mL human FGF-2 (PeproTech), 1 × B27 supplement (Invitrogen), 0.5 × N2 supplement (Invitrogen), 2 mg/mL heparin (Sigma), and 0.5 × penicillin G-streptomycin sulfateamphotericin B complex (Mediatech). For passaging, spheres were centrifuged, treated with Accutase (Innovative Cell Technologies) at 37°C for 5 min, dissociated by pipetting, washed with PBS, and resuspended in EF20 medium [13].…”

“…The expression of Notch-1 (1:150, Santa Cruz Biotechnology) was evaluated by immunofluorescent staining according to the manufacturer's instructions. Sections were examined for positive staining that was quantified as previously described [13].…”

Inhibition of Notch pathway enhances the anti-tumor effect of docetaxel in prostate cancer stem-like cells

“…(1:150, Santa Cruz Biotechnology) was evaluated by immunofluorescent staining according to the manufacturer's instructions. Sections were examined for positive staining that was quantified as previously described [13].…”

Inhibition of Notch pathway enhances the anti-tumor effect of docetaxel in prostate cancer stem-like cells

“…PCSCs were cultured in EF20 medium composed of Neurobasal medium (Invitrogen) supplemented with 3 mM L-Glutamine (Mediatech), 20 ng/mL human EGF (R&D Systems), 20 ng/mL human FGF-2 (PeproTech), 1 × B27 supplement (Invitrogen), 0.5 × N2 supplement (Invitrogen), 2 mg/mL heparin (Sigma), and 0.5 × penicillin G-streptomycin sulfate-amphotericin B complex (Mediatech). For passaging, spheres were centrifuged, treated with Accutase (Innovative Cell Technologies) at 37℃ for 5 min, dissociated by pipetting, washed with PBS, and resuspended in EF20 medium [13].…”

“…The expression of Notch-1 (1:150, Santa Cruz Biotechnology) was evaluated by immuno uorescent staining according to the manufacturer's instructions. Sections were examined for positive staining that was quanti ed as previously described [13].…”