B66
Detection of Akt activation in early breast cancer using proximity ligation assays
Melanie Spears1, Carrie Cunningham1, Elizabeth Mallon2, John Bartlett1
1University of Edinburgh, UK, 2Western Infirmary, Glasgow, UK
Background
The Akt family of kinases play an important role in cancer progression and cell survival. They are activated by a variety of stimuli through growth factor receptors in a PI3K-dependent manner. There a three isoforms, Akt1, Akt2 and Akt3. The activation of Akt has been shown to be associated with a worse outcome among endocrine-treated breast cancer patients. Akt phosphorylation is associated with resistance to tamoxifen and doxorubicin. High tumours levels of cytoplasmic Akt2 are associated with improved overall survival. Using breast cancer and the Akt family of proteins as a model system we demonstrate, for the first time, in situ detection of isoform specific activation of Akt1 and Akt2 in both cultured cells and formalin fixed breast cancer samples.
Method
A panel of breast cancer cells lines and 100 early breast cancer specimens were used to detect phosphorylated expression of Akt (Thr 308), Akt1, and Akt2 through IHC and western blotting. Isoform specific activation was detected by proximity ligation assay (PLA).
Results
Western blot analysis confirmed a range of Akt expression. Akt activation signals were detected in all cases with a range of signals observed. Cell lines that had high Akt expression levels by western blotting showed a high levels of Akt activation signals, whereas, cell lines that had minimal to no Akt expression had very little signal. IHC and PLA demonstrated a range of expression in breast cancer cases. The was a high inter-run correlation for the PLA experiments (Akt1:pAkt = 0.9 and Akt 2:pAkt = 0.7). The mean number of Akt1:pAkt signals (7 signals per cell) was greater than Akt2:pAkt (3 signals per cell) in this series indicating that Akt1 is the predominant activator.
Conclusion
We have demonstrated that PLA is an invaluable method to detect isoform specific activation of Akt in breast cancer samples.
