Abstract:

PIK3CA mutations are critical drivers of osteosarcoma progression, yet their exact molecular circuitry, functional mechanisms, and therapeutic responsiveness remain incompletely understood. In this study, we overexpressed wild-type and mutant PIK3CA genes in cell models, and identified the differences between the adjacent proteomes by the labelling technology to further analyse the proximity protein networks in the PI3K pathway. The results showed that the cancer cells carrying PIK3CA mutations established a unique protein network composed of different proteins, distinct from those carrying wild-type PIK3CA. By covalently labelling adjacent proteins in HEK293FT cells and analysing them via mass spectrometry, we discovered that the two hotspot oncogenic mutants of PIK3CA, H1047R and E545K, exhibit differential regulation in their interactions with the aldolase. The PIK3CA[E545K] mutant exhibited specific interactions with TUBA3C, B4DQK4, Q8N9M2, AHSA1, ELAVL1, C1QBP and DDX41, whereas the PIK3CA[H1047R] mutant was uniquely associated with proximal proteins including MDH2, RBBP6, RPL10, RPS5, SSBP1 and NOLC1. Furthermore, mass spectrometry analysis revealed that SLBP and DKC1 within the CDK2 pathway were downregulated in both H1047R and E545K mutants. In contrast, EEF2, CCT6A and PPIA from the HSP90 pathway, together with IGF2BP1 in the PI3K pathway, were upregulated. In summary, these findings provide a detailed elucidation of the complexity of the oncogenic PIK3CA molecular networks, offering valuable guidance for the development of inhibitors or other therapeutic approaches, as well as important insights into the mechanisms of tumorigenesis.