Proteomic alterations in extracellular vesicles induced by oncogenic PIK3CA mutations

Abstract

PIK3CA is one of the most frequently mutated genes in human cancers, with the two most prevalent activating mutations being E545K and H1047R. Although the altered intracellular signaling pathways in these cells have been described, the effect of these mutations on their extracellular vesicles (EVs) has not yet been reported. To study altered cellular physiology and intercellular communication through proteomic analysis of EVs, MCF10A cells and their isogenic mutant versions (PIK3CA E545K and H1047R) were cultured and their EVs enriched by differential ultracentrifugation. Proteins were extracted, digested with trypsin and the peptides labeled with tandem mass tag (TMT) reagents and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Four thousand six hundred and fifty-five peptides were identified from 579 proteins of which 522 proteins have been previously described in EVs. Relative quantitation revealed altered levels of EV proteins including several cell adhesion molecules. Mesothelin, E-cadherin, and epithelial cell adhesion molecule were elevated in both mutant cell-derived EVs. Markers of tumor invasion and progression like galectin-3 and transforming growth factor beta induced protein were increased in both mutants. Overall, activating mutations in PIK3CA result in altered EV composition with characteristic changes associated with these hotspot mutations.

The 2022 Report on the Human Proteome from the HUPO Human Proteome Project

“…92 Extracellular vesicles, small membrane-enclosed particles released by cells and able to traffic information between them, are seeing increasing application, for example, proteomic alterations in extracellular vesicles from activating mutations in PIK3CA, one of the most frequently mutated genes in human cancers. 93 At the technical level, a surfactant-assisted one-pot sample preparation for label-free single-cell proteomics improves detection sensitivity for single-cell proteomics 94 to address intratumoral heterogeneity. There were also reviews on proteomics, personalized medicine, and cancers, 85,95 and roles of separation sciences in proteomics.…”

“…Martens et al developed a molecular pathway model of malignant pleural mesothelioma (MPM), a visual, interactive overview of interactions and connections between proteins and molecular pathways known to be related to MPM . Extracellular vesicles, small membrane-enclosed particles released by cells and able to traffic information between them, are seeing increasing application, for example, proteomic alterations in extracellular vesicles from activating mutations in PIK3CA, one of the most frequently mutated genes in human cancers . At the technical level, a surfactant-assisted one-pot sample preparation for label-free single-cell proteomics improves detection sensitivity for single-cell proteomics to address intratumoral heterogeneity.…”

“…The Project includes the following aspects: Quantitative status of proteins (expression level, isoforms, PTMs, localization, interactions) in the context of networks and interactomes that can be perturbed by variation among individuals, specific binding interactions, and protein complex formation, influencing their functional relevance. Computational predictions , may provide Gene Ontology clues for experimental design. Measurements of proteins can be based on MS, antibodies, and any other technologies with rigorous technical quality control.…”