Therapeutic Modulation of Akt Activity and Antitumor Effi cacy of Interleukin-12 Against Orthotopic Murine Neuroblastoma

Background: Patients with advanced neuroblastoma have a poor prognosis. The antiapoptotic protein Akt has been implicated as a possible mediator of the resistance of human neuroblastoma cells to apoptosis; the proapoptotic protein Bid, is inhibited by activated Akt. Neuroblastoma has dem- onstrated responsiveness to immunotherapeutic approaches in preclinical studies, prompting investigation of new thera- peutic strategies based on potentiation of the host immune response, including the use of systemic cytokines. Methods: We examined the antitumor effi cacy and mechanisms of action of the central immunoregulatory cytokine interleukin- 12 (IL-12) in mice bearing established orthotopic neuroblas- toma tumors derived from murine TBJ and Neuro-2a cells. Cohorts of mice (10 mice/group) bearing established ortho- topic neuroblastoma tumors were injected intraperitoneally with IL-12 or vehicle and monitored for survival. IL-12 - induced apoptosis within the tumor microenvironment was investigated using ribonuclease protection assays, nuclear staining, and electron microscopy. Protein expression was de termined via Western blot analysis and enzyme-linked immunosorbent assays. Confocal microscopy was used to examine the distribution of overexpressed Bid - enhanced green fl uorescent protein fusion protein (Bid-EGFP) in TBJ cells. All statistical tests were two-sided. Results : IL-12 induced complete tumor regression and long-term survival of 8 (80%) of 10 mice bearing established neuroblastoma tumors compared with 1 (10%) of 10 control mice ( P = .0055) and profound tumor cell apoptosis in vivo despite the fact that TBJ and Neuro-2a cells were resistant to receptor- mediated apoptosis in vitro . These cells expressed high levels of phos- phorylated Akt, a key prosurvival molecule, and Akt inhibi- tors sensitized neuroblastoma cells to apoptosis mediated by IL-12 - inducible cytokines including tumor necrosis factor- α and interferon- γ in vitro. IL-12 increased the expression of proapoptotic genes and decreased Akt phosphorylation within established TBJ tumors in conjunction with activation and subcellular translocation of Bid. Conclusions: Our results suggest that IL-12 overcomes a potentially critical mecha- nism of tumor self-defense in vivo by inhibiting Akt activity and imply that IL-12 may possess unique therapeutic activity against tumors that express high levels of activated Akt. (J Natl Cancer Inst 2006;98:190 - 202)