Abstract Mutations and amplification in genes regulating the PI 3-K/Akt pathway are common in prostate cancer, with deletion or mutations of PTEN being the most frequent. This has led to major efforts in developing anti-cancer drugs targeting the Akt pathway. Although the role of Akt in tumorigenesis has been studied extensively, little is known regarding isoform-specificity, and it is largely assumed that Akt isoforms play redundant roles in regulating cancer cell survival. In this study, we have developed a tet-on inducible system for expressing Akt1/2/3 shRNAs to determine the role of Akt isoforms in prostate cancer progression. Three-dimensional (3D) matrigel cultures are employed to better recapitulate tumors growing in vivo. Administration of doxycycline (dox) in Akt1 or Akt2 shRNA PTEN-deficient LNCaP cells at the start of culture resulted in the inhibition of spheroid formation, suggesting an important role of both Akt1 and Akt2 in the initial growth of spheroids. Conversely, when spheroids were allowed to form for 7 days, followed by dox for further 10 days, no difference was observed between Akt1-depleted and control spheroids. In contrast, silencing of Akt2 resulted in complete disintegration of spheroids. Confocal microscopy studies showed a significant induction of active caspase-3 and fragmented nuclei in Akt2-silenced cells. Moreover, whereas Akt1 is uniformly expressed, Akt2 is localized in the cytoplasm and/or on the plasma membrane. Interestingly, the differences of effect of Akt isoforms on cell survival in conventional 2D culture are considerably more modest. The exclusive requirement of Akt2 for survival is also observed in PC3 prostate cancer cells, which are also PTEN-deficient. Importantly, our preliminary data indicated that Akt2, but not Akt1 knockdown, led to tumor regression in the LnCaP xenograft model. These results indicate that Akt2, but not Akt1, plays a critical role in prostate cancer maintenance. Current efforts are aimed at identifying novel Akt isoform-specific substrates that contribute to prostate cancer progression using a genome-wide proteomic screen. The results of these studies could point to new targets for anti-cancer therapeutics. This work was supported by grants from NIH (A.T., CA122099; R.C., 1K99CA157945-01, T32 CA081156-09) Citation Format: Yuet Ming Rebecca Chin, Steve Balk, Alex Toker. Akt2-specific signaling in prostate cancer maintenance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4090. doi:10.1158/1538-7445.AM2013-4090