Abstract 985: Dissecting PI3K-AKT pathway by phosphoproteomic profiling of PIK3CA knock-in mutants

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The phosphatidylinositol 3-kinase subunit PIK3CA is frequently mutated in human cancers. PIK3CA mutations are observed in about 30% of breast cancers. Three recurrent oncogenic “hotspot” mutations comprise the majority of somatic PIK3CA mutations. Two of these mutations, E542K and E545K, occur in the helical domain found in exon 9, and the third mutation, H1047R, affects the kinase domain located within exon 20. Although many studies have implicated PIK3CA mutations with features of transformation, definitive mechanisms describing how these mutations promote cell growth and proliferation have not been fully elucidated. Two MCF10A (a normal mammary gland epithelial cell line) knock-in cell lines with E545K or H1047R hotspot mutations were used for our phosphoproteome study. We employed stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative mass spectrometry to study phosphoproteomic alterations caused by PIK3CA onogenic mutations. In order to identify signaling proteins directly involved in PI3K-AKT pathway, a novel PIK3CA specific inhibitor (J124) was used to suppress PIK3CA activity. Phosphopeptides were enriched by TiO2-based strategy after strong cation exchange (SCX) fractionation and analyzed on a high resolution Fourier transform LTQ-Orbitrap Velos mass spectrometer. We identified 5,491 unique phosphopeptides from 1,546 unique proteins - of these, 2,239 peptides were hyperphosphorylated in MCF10A cell with PIK3CA mutants. Notably, the PIK3CA inhibitor, J124, could specifically suppress the phosphorylation levels of 1,187 hyperphosphorylated peptides. These are likely to be effectors of PI3Kalthough only 34 of them were previously known AKT1 substrates. By integrating data from protein microarray-based phosphorylation experiments with AKT1, we found 16 novel hyperphosphorylated proteins that were phosphorylated in vitro by AKT1. In addition to identifying novel AKT substrates, we also identified multiple receptor tyrosine kinases including EGFR, FGFR and EPHA2 to be hyperphosphorylated in PIK3CA mutant knockin cells. This novel discovery of the reverse crosstalk initiated from PI3K-AKT to RTKs could explain oncogenic consequences of cells with PIK3CA mutants such as growth factor-independent growth and invasive ability. In summary, our comprehensive phosphoproteomic study reveals that oncogenic activation of the PI3K-AKT pathway can activate a broad spectrum of signaling molecules. By integrating data from large-scale protein microarray studies, we were able to identify novel AKT substrates. The knowledge gained from these studiescan greatly enhance our understanding of the PI3K-AKT pathway and provide potential therapeutic targets for attenuating oncogenic effects of PIK3CA mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 985. doi:1538-7445.AM2012-985