Abstract 3078: PHLPP1 phosphatase dephosphorylates Akt and inhibits tumorogenesis in the orthotopic model of pancreatic cancer
Authors: Claudia J. Nitsche, Mouad M. Edderkaoui, Guido S. Eibl, Noriyuki Kasahara, Paul Grippo, Julia Mayerle, Markus M. Lerch, Anna S. Gukovskaya
Journal: Cancer Research (2010)

Abstract Background &amp; Aims: Akt kinase is a major prosurvival factor in pancreatic cancer (PaCa). Akt activity is regulated by its phosphorylation, which is controlled by kinases and phosphatases. Whereas kinases involved in Akt phosphorylation are well characterized, little is known about phosphatases that inactivate Akt. Here we investigated the expression of recently discovered serine phosphatase PHLPP1 in human and mouse pancreatic adenocarcinomas; its role in the regulation of Akt activity and PaCa cell death; and the effect of PHLPP1 overexpression on tumorigenesis in the nude mouse orthotopic model of PaCa. Methods: Human pancreatic adenocarcinoma MiaPaCa-2 and PANC-1 cells were cultured in the presence or absence of insulin-like growth factor-I (IGF-I) or serum (FBS). We measured apoptosis by DNA fragmentation and AnnexinV staining (flow cytometry); Akt activity, by Akt phosphorylation at Ser473; and phosphatase activity in PHLPP1 immunoprecipitate. For transient overexpression PHLPP plasmid were transfected using Amaxa nucleofector. MiaPaCa-2 cells transduced with PHLPP1 expressing lentivirus (as well as control virus) were applied in the orthotopic model of PaCa. PDXCre Kras p53 mice were used as a transgenic pancreatic cancer model. Results: Overexpression of PHLPP1 in PaCa cells reduced, whereas its’ knock-down with siRNA increased Akt phosphorylation. Accordingly, PHLPP1 overexpression increased, whereas its’ knock-down decreased apoptosis in PaCa cells. PHLPP1 stimulated death of PaCa cells through inhibiting Akt. Immunofluorescence showed that PHLPP 1 expression in human and mouse pancreatic adenocarcinomas was significantly lower than in normal pancreas. PHLPP1 activity in PaCa cells was strongly inhibited by NADPH oxidase Nox4, which is highly expressed in pancreatic cancer and is co-localized with PHLPP1. Furthermore, the activity of PHLPP1 in PaCa cells was inhibited by the growth factor IGF-I, which is abundant in pancreatic tumors. This inactivation is at least partly due to ROS since inhibition of IGF-I induced ROS production increased PHLPP1 activity in vivo and peroxide greatly inhbited PHLPP1 activity in vitro. PHLPP 1 overexpression greatly delayed subcutaneous tumor induction and decreased by 2.75± 0.8 (n=6) times tumor volume in orthotopic pancreatic cancer model. Conclusion: The data indicate that phosphatase PHLPP1 is a key negative regulator of Akt in PaCa cells. The level and activity of PHLPP1 drastically decreased in pancreatic cancer resulting in high Akt phosphorylation and the inhibition of PaCa cell death. Overexpression of PHLPP1 inhibits tumorogenesis in the orthotopic model of pancreatic cancer demonstrating that PHLPP1 is a promising target for treatment of pancreatic adenocarcinoma. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3078.