Global Analysis of the PI3K Signaling Pathway

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

Correction to: Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts

Unfortunately, after publication of this article [1], errors were noticed in Figs. 3 and 4

Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts

Abstract Background Cancer-associated fibroblasts (CAFs) are one of the most important components of tumor stroma and play a key role in modulating tumor growth. However, a mechanistic understanding of how CAFs communicate with tumor cells to promote their proliferation and invasion is far from complete. A major reason for this is that most current techniques and model systems do not capture the complexity of signal transduction that occurs between CAFs and tumor cells. Methods In this study, we employed a stable isotope labeling with amino acids in cell culture (SILAC) strategy to label invasive breast cancer cells, MDA-MB-231, and breast cancer patient-derived CAF cells. We used an antibody-based phosphotyrosine peptide enrichment method coupled to LC–MS/MS to catalog and quantify tyrosine phosphorylation-mediated signal transduction events induced by the bidirectional communication between patient-derived CAFs and tumor cells. Results We discovered that distinct signaling events were activated in CAFs and in tumor epithelial cells during the crosstalk between these two cell types. We identified reciprocal activation of a number of receptor tyrosine kinases including EGFR, FGFR1 and EPHA2 induced by this bidirectional communication. Conclusions Our study not only provides insights into the mechanisms of the interaction between CAFs and tumor cells, but the model system described here could be used as a prototype for analysis of intercellular communication in many different tumor microenvironments

Activating Mutations in <i>PIK3CA</i> Lead to Widespread Modulation of the Tyrosine Phosphoproteome

The human oncogene <i>PIK3CA</i> is frequently mutated in human cancers. Two hotspot mutations in <i>PIK3CA</i>, E545K and H1047R, have been shown to regulate widespread signaling events downstream of AKT, leading to increased cell proliferation, growth, survival, and motility. We used quantitative mass spectrometry to profile the global phosphotyrosine proteome of isogenic knock-in cell lines containing these activating mutations, where we identified 824 unique phosphopeptides. Although it is well understood that these mutations result in hyperactivation of the serine/threonine kinase AKT, we found a surprisingly 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 that there is frequent and unexpected cross-talk that occurs between tyrosine signaling pathways and serine/threonine signaling pathways activated by the canonical PI3K-AKT axis