 Hello, my name is Bedour Selhia, and I am a research investigator at the Translational Genomics Research Institute in Phoenix, Arizona. Today I will be talking to you about a paper that I am the lead author on that recently got published in the journal Human Mutation. It is entitled Differential Effects of AKT-1 E17K Expression on Human Mammary Luminal Epithelial and Miloepithelial Cells. This work began as a result of the discovery made by the current paper's senior author, Dr. John Carpton and colleagues, identifying the very first mutation in the AKT-1 gene. The work was published in the journal Nature in 2007. The mutation is characterized by a glutamic acid to lysine substitution in the 17th amino acid position of the plexron-homology domain. So the purpose of the current study was to understand the pathobiological role of the E17K mutation in human mammary epithelial cells. To study the functional significance of the E17K mutation in human mammary epithelial cells, we used the genetically matched cell line model derived from both non-tomogenic and transformed miloepithelial-like and luminal-like precursors. The non-tomogenic cells are referred to as HME and BPE cells, and the transformed derivatives are referred to as Hemler and Butler cells. So like the previous report, we did confirm that E17K was consistently active in the human mammary epithelial cells as well. And this was known or shown by virtue of hyperphosphorylation of the regulatory amino acids, plasma membrane localization, and hyperphosphorylation of 4-ket transcription factors. We also demonstrated that in spite of constitutive activity, E17K was responsive to the PI3 kinase inhibitor LI294002, suggesting still that there is a dependence on PI3 kinase. We provide a possible explanation for this phenomenon in our manuscript, but we do believe it may be related to the crystal structure of E17K. Among the functionally intriguing findings in our study was the inhibition of cell growth by E17K in all four mammary epithelial cell lines. This suggested to us that E17K cannot be assumed to be oncogenic in all cell types. Interestingly, also found resistance to enhanced resistance to various cytotoxic agents in the luminal setting by E17K cells. We believe this could have important clinical implications and is discussed further in our manuscript. Our differential proteomics analysis in HME and BPE cells did demonstrate unique differences between E17K and wild-type AKT1. We also showed a strong propensity for differentially expressed proteins to be downregulated in each 17K cells. My colleague at Teej and Dr. Kostas Petritens, who is also co-author on the paper, will now describe the differential proteomics approach we used in our study. I'm Kostas Petritis, I'm the laboratory head of the Center for Proteomics Alliance. In three different conditions, we took the cells, inaturated, reduced and alkylate, digested with trypsin, labelled with TMT, and then analyzed with a mass spectrometer. And this gives us a lot of raw data that needs to be normalized, which we do with in-house software. Here you can see after normalization. And with this method, you can identify thousands of proteins. In this case, we have more than 1500 proteins that we identified. And then with further filtering, we ended up with about 470 proteins. And you can see in this heat map proteins overexpressed or underexpressed other different conditions. In summary, our study illustrates the importance of cellular context in understanding the biological and clinical relevance of putative oncogenic mutations like the AK-T1E17K breast cancer. We believe, though, that this mutation may be ultimately associated with favorable prognosis. And so determining whether the use of PI3 kinase inhibitors or AK-T inhibitors in patients with a mutation would be an effective strategy or not will be an important next step. We hope our video now entices you to read our manuscript, now published in Human Mutation. We thank you very much for your time and interest. And if you have any further questions, please do not hesitate to contact us.