BACKGROUND & AIMS The kinase Akt mediates resistance of pancreatic cancer (PaCa) cells to death and is constitutively active (phosphorylated) in cancer cells. Whereas the kinases that activate Akt are well characterized, less is known about phosphatases that dephosporylate and thereby inactivate it. We investigated regulation of Akt activity and cell death by the phosphatases PHLPP1 and PHLPP2 in PaCa cells, mouse models of PaCa, and human pancreatic ductal adenocarcinoma (PDAC). METHODS We measured the effects of PHLPP overexpression or knockdown with small interfering RNAs on Akt activation and cell death. We examined regulation of PHLPPs by growth factors and reactive oxygen species, as well as associations between PHLPPs and tumorigenesis. RESULTS PHLPP overexpression inactivated Akt, whereas PHLPP knockdown increased phosphorylation of Akt in PaCa cells. Levels of PHLPPs were greatly reduced in human PDAC and in mouse genetic and xenograft models of PaCa. PHLPP activities in PaCa cells were down-regulated by growth factors and Nox4 reduced nicotinamide adenine dinucleotide phosphate oxidase. PHLPP1 selectively dephosphorylated Akt2, whereas PHLPP2 selectively dephosphorylated Akt1. Akt2, but not Akt1, was up-regulated in PDAC, and Akt2 levels correlated with mortality. Consistent with these results, high levels of PHLPP1, which dephosphorylates Akt2 (but not PHLPP2, which dephosphorylates Akt1), correlated with longer survival times of patients with PDAC. In mice, xenograft tumors derived from PaCa cells that overexpress PHLPP1 (but not PHLPP2) had inactivated Akt, greater extent of apoptosis, and smaller size. CONCLUSIONS PHLPP1 has tumor suppressive activity and might represent a therapeutic or diagnostic tool for PDAC.

Abstract Background & Aims: Autophagy plays a dual role in cancer development. Recent data indicate that autophagy is oncosuppressive and impaired autophagy is emerging as the critical pro-onco “hallmark” of cancer cells. On the other hand, autophagy is a prosurvival mechanism, and its inhibition may increase cancer cell death and thus be beneficial. Beclin-1 plays a key role in mediating autophagy; its interaction with class III PI3K hVPS34 was shown to be necessary for the autophagosome formation in eukaryotic cells and Beclin-1 knockdown promotes tumorigenesis. Here we investigate the role of Beclin-1 in autophagy and apoptosis in pancreatic cancer cells. Methods: Pancreatic cancer (PaCa) cells PANC-1, and CAPAN-2 were subjected to amino-acid (AA) depletion or hypoxia, strong autophagy activators. Measuring autophagic marker LC3-II with western blot or with immunofluorescence assessed the number of autophagic vacuoles. Autophagy induction was measured in cells pretreated with NH4Cl or bafilomycin to block autophagy progression. Fusion of autophagosomes with lysosomes was assessed by co-localization of lysosomal marker LAMP-1 with LC3; the efficiency of autophagic flux by measuring the levels of p62, which is degraded through autophagy; apoptosis by DNA fragmentation, and caspase-3 activity. Results: AA depletion and hypoxia increased LC3-II both in the presence and absence of NH4Cl, indicating autophagy induction. AA depletion and hypoxia decreased p62 level, indicating stimulation of autophagic degradation. In cells treated with or without AA depletion or hypoxia Beclin-1 knockdown had no effect on either autophagic vacuole formation or p62 degradation. Similarly, the inhibition of Beclin-1 partner, hVPS 34 with siRNA or with PI3K inhibitor 3-methyladenine (3MA), decreased neither autophagy induction nor autophagic degradation. Finally, silencing of both Beclin-1 and its downstream mediator, Atg5 did not affect autophagy induction by AA depletion or hypoxia. All these data indicate that autophagy in PaCa cells is mediated through Beclin-1, Atg5, hVPS 34 -independent pathway. Of note, Beclin-1 knockdown increased co-localization of LC3 with LAMP-1 and p62 levels indicating that autophagy progression is delayed in the absence of Beclin-1. AA depletion and hypoxia moderately (∼by 30%) increased apoptosis in PaCa cells. Silencing of Beclin-1, hVPS 34, or Atg5 had no effect on apoptosis, in basal conditions as well as well as in cells subjected to AA depletion or hypoxia. Differently, 3MA stimulated apoptosis through Beclin-1-independent mechanism. Conclusions: In pancreatic cancer cells AA depletion and hypoxia activate autophagy through non-canonical pathway, which does not involve Beclin-1. Silencing of the key mediators of canonical autophagy, Beclin-1, Atg5 and hVPS34 had no effect on autophagy and apoptosis, whereas 3MA stimulated apoptosis through autophagy-independent pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3775. doi:10.1158/1538-7445.AM2011-3775

Background & Aims: Key steps in autophagic protein degradation are formation of autophagosomes (induction) and their fusion with lysosomes (progression). Akt is a major anti-apoptotic factor in cancer cells; however, its role in autophagy in general, and in pancreatic cancer (PaCa) in particular, is poorly understood. We recently showed that phosphatases PHLPP-I and -II are key negative regulators of Akt in PaCa cells. The present study aimed to elucidate the effects of Akt and PHLPPs on individual steps of autophagy and the role of autophagy in the prosurvival function of Akt in PaCa cells. Methods: We measured apoptosis by DNA fragmentation and caspase-3 activity; autophagy, by conversion of cytosolic LC3-I to autophagic LC3-II (with immunoblot) and LC3 immunofluorescence; autophagy induction, in cells pretreated with NH4Cl to block autophagy progression; autophagosomal fusion with lysosomes, by co-localization of the lysosomal marker LAMP-1 with LC3-II; and efficiency of protein degradation, by level of p62 protein, a specific autophagy target. Results: Akt inactivation by AktVIII inhibitor or siRNA, or by overexpression of PHLPP-I and -II all stimulated autophagic vacuole formation in MiaPaCa-2 and PANC-1 cells. Akt inactivation greatly facilitated LC3-I/LC3-II conversion both in the presence and absence of NH4Cl, indicating increased autophagy induction. At the same time, Akt inactivation inhibited autophagosomal fusion with lysosomes and efficiency of protein degradation, indicating decreased autophagy progression. Thus, Akt has dual effect on autophagy in PaCa cells: it inhibits autophagy induction but promotes autophagosomal fusion with lysosomes and hence autophagy progression. The overall effect of Akt is decreased accumulation of autophagic vacuoles, i.e., inhibition of autophagic death. To study the role of autophagy in apoptosis, we used 3-methyladenine or beclin siRNA to inhibit autophagy induction, and bafilomycin or chloroquine to block the fusion of autophagosomes with lysosomes. All these treatments stimulated apoptosis in PaCa cells, indicating the anti-apoptotic role of autophagy. In cells with inactivated Akt, blocking autophagy induction (e.g., with 3-methyladenine) further greatly stimulated apoptosis. This indicates that the ability of Akt to inhibit autophagy induction greatly diminishes its anti-apoptotic function. Differently, blocking autophagosomal fusion with lysosomes had little effect on apoptosis in cells in which Akt was inactivated, likely because in these cells autophagy progression was already inhibited. Conclusions: Akt inhibition hampers autophagy progression, thus promoting vacuolization and autophagic cell death. At the same time, Akt inhibition greatly stimulates autophagy induction, thus counteracting apoptosis. Therefore, to maximally stimulate apoptosis in PaCa cells we propose to apply Akt inhibitors in combination with inhibitors of autophagy. 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 4844.