Abstract 4046: Direct detection of AKT/PKB activity in a Pten knock out mouse model using dynamic peptide microarrays
Authors: Hilhorst Maria H. , Houkes Liesbeth , Korsten Hanneke , Mommersteeg Monique , Trapman Jan , Ruijtenbeek Rob 
Published: August 2014

Abstract
Background. In many cancers, deregulation of kinases or phosphatases leads to activation of the AKT/PKB pathway. This pathway regulates many cellular processes including metabolism, differentiation, translation, proliferation and apoptosis, so is central in determining cell fate. Several kinase inhibitors that influence AKT activity are investigated in the context of new pharmacotherapies in cancer. However, often a fraction of patients responds and it is not possible to identify those patients up front. It would be a major leap forward if response to therapy could be determined in an in vitro assay prior to the onset of therapy. Methods to directly measure this activity do not exist.
In this study, we explored:
1) Whether it is feasible to determine AKT activity profiles in lysates of hyperplastic prostates of PSA-Cre;Pten-loxP/loxP mice.
2) Whether addition of AKT inhibitors affects the phosphorylation profiles in an inhibitor specific way.
Methods. The PSA-Cre;Pten-loxP/loxP mouse prostate cancer model was described in Ma et al, 2005 Cancer Res 65 5730 and Korsten et al, 2009 PLoS ONE 4(5) e5662. Prostate tissues of three 4 months old PSA-Cre;Pten-loxP/loxP mice and three wt mice were lysed in buffer with protease and phosphatase inhibitors. Lysates and recombinant AKT were incubated in triplicate on PamChip® peptide micro-arrays (Folkvord et al. Int J Radiat Oncol Biol Phys. 2010 78:55) comprising 140 or 256 ser/thr containing peptides derived from known human phosphorylation sites. Peptide phosphorylation was monitored with anti- phosphoserine and anti-phosphothreonine antibodies. Incubations were performed in the presence and absence of AKT inhibitors MK2206 and AKT Inhibitor VIII. Western blotting was performed using standard methods. Blots were stained with anti-phospho-AKT antibodies and anti total AKT antibody.
Results. Phosphorylation activity profiles revealed differential activity between PSA-Cre;Pten-loxP/loxP and wt mouse prostate lysates, with the Pten negative lysate having increased activity. Addition of AKT inhibitors reduced signals on several of these peptides in the prostate lysates from targeted Pten knockout mice, but not in the lysates of the wt mice. Addition of non-AKT specific inhibitors like UCN-01 resulted in signal reduction in both lysates.
Western blots showed the presence of AKT in the wt lysate and in the Pten knockout lysate. Only the Pten knockout lysates contained AKT phosphorylated on S473 and T308.
Conclusion. In PSA-Cre;Pten-loxP/loxP mouse prostate tissue lysate, the activity of AKT is increased compared to wild type tissue levels. The activity profiles of the PTEN/AKT pathway will support the studies on the complicated regulation of AKT activity. Up to now these studies were limited to detection of phosphoprotein abundances. This new multiplex kinase activity assay will add a new dimension and a scope of applications like drug testing.
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 4046. doi:10.1158/1538-7445.AM2011-4046