Abstract 3775: Autophagy in pancreatic cancer cells is mediated through a non-canonical Beclin-1 independent pathway

• Published in: Cancer research (Chicago, Ill.) Ill.), 2011-04, Vol.71 (8_Supplement), p.3775-3775
• Main Authors: Nitsche, Claudia J., Moore, Ryan M., Maertin, Sandrina, Mayerle, Julia, Lerch, Markus M., Grippo, Paul, Gukovskaya, Anna S.
• Format: Article
• Language: English
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2011-3775

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 hy