Abstract 1060: A functional and proteomics analysis of AKT1(E17K) in human mammary epithelial cells of luminal and myoepithelial cells of origin

Abstract

Abstract The normal breast epithelium, from which most breast cancers arise, is composed of an inner layer of luminal cells and an outer layer of myoepithelial (basal) cells. A cell culture method recently described allows direct comparison of two genetically matched cells derived from normal myoepithelial and luminal epithelial cell types termed HME and BPE cells respectively (Ince et al, Cancer Cell 2007). Transformation of these cells (subsequently termed HMLER and BPLER) formed tumor xenografts exhibiting major differences in histopathology, tumorigenicity, and metastatic behavior. The non-receptor serine/threonine kinase AKT/PKB is one of the most commonly deregulated pathways in human breast cancer. In this regard we performed functional and proteomics assays to compare the role of wild-type (wt) AKT1 versus the naturally occurring AKT1 mutant (E17K), in human breast cancer. We demonstrated that BPE and HME cells expressing AKT1(E17K) have constitutive plasma membrane localization and hyper-phosphorylation at serine-473 and threonine-308, demonstrating that activation of E17K is growth-factor independent. However, additional studies suggest that HME and BPE cells expressing wt and mutant AKT1 have differing activation kinetics, the reasons of which are unknown. Using a sulforhodamine B and transwell migration assays we demonstrated that E17K inhibits cell growth and migration respectively in BPE and HME cells. However, E17K enhances chemoresistance compared with wt AKT1 function. Interestingly, while E17K inhibits migration in HMLER cells it leads to increased cellular migration in BPLER cells. In order to investigate the molecular basis of AKT1(E17K) function in the different epithelial cell lineages, we performed differential proteomics analysis using liquid chromatography (LC) mass spectrometry (MS) analysis. Although several LCMS methods allowing the characterization and quantification of proteins have been developed we chose two unique styles using 2-dimensional reverse phase reverse phase LC for both. The first method is label-free quantitation utilizing spectral counting. This technique counts the number of spectra identified for a given peptide in each sample and results integrated for all peptides detected for a given protein. The second being iTRAQ, an isobaric labeling technique that uses isotope coded covalent tags for relative quantitation. Using these approaches we identified several hundred differentially expressed proteins between wt and mutant AKT1 and between HME and BPE cells. The proteins from our analysis converged on various cellular functions including pathways underlying cellular migration. Overall the data leads us towards understanding the molecular mechanisms that discriminate between mutant and wt AKT1 function and demonstrates the importance of cellular context in determining genotypic effects of oncogenic mutations. 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 1060. doi:10.1158/1538-7445.AM2011-1060