Global Analysis of the PI3K Signaling Pathway

Abstract

The human oncogene PIK3CA is frequently mutated in human cancers. Two hotspot mutations in PIK3CA, E545K and H1047R, have been shown to regulate widespread signaling events downstream of AKT, leading to increased cell proliferation, growth, survival, and motility. Although many studies have associated PIK3CA mutations with features of transformation, a global and quantitative study of how mutant PIK3CA impacts the signaling networks and consequently transforms epithelial cells has not yet been described. The goal for this thesis project was to systematically dissect the signaling pathways that are activated due to these PIK3CA mutations in a global manner utilizing the power of phosphoproteomics and mass spectrometry. To this end, we employed stable isotope labeling of amino acids in cell culture (SILAC) to precisely identify and quantify the phosphorylation changes that occur in an isogenic series of immortalized non-tumorigenic breast epithelial cell lines containing E545K and H1047R mutations. We performed two phosphopeptide enrichment methods, namely titanium dioxide (TiO2) beads to enrich for mainly serine/threonine phosphorylated peptides and anti-phosphotyrosine antibody to enrich for tyrosine phosphorylated peptides followed by high resolution LC-MS/MS analysis. From ~9000 unique phosphopeptides identified, we found that aberrant activation of PI3K pathway leads to increased phosphorylation of a surprisingly wide variety of kinases and downstream signaling networks. By integrating the phosphoproteomic data with human microarray-based AKT1 kinase assays, we discovered and validated six novel AKT1 substrates. One of these substrates, cortactin, was found to be important in conferring the cells with invasion/migration advantage. Through mutagenesis studies, we demonstrated that phosphorylation of cortactin by AKT1 is important for mutant PI3K-enhanced cell migration and invasion. Although it is well understood that these mutations in PIK3CA result in hyperactivation of the serine/threonine kinase AKT, we also observed an unexpected widespread modulation of tyrosine phosphorylation levels of proteins in the mutant cells. In the tyrosine kinome alone, 29 tyrosine kinases were altered in their phosphorylation status. Many of the regulated phosphosites that we identified were located in the kinase domain or the canonical activation sites, indicating that these kinases and their downstream signaling pathways were activated. Our study demonstrates the utility of a quantitative and global approach to identify mutation-specific signaling events and to discover novel signaling molecules as readouts of pathway activation or potential therapeutic targets.