(Phospho)proteomic Profiling Reveals Mutation-Specific Adaptive Signaling to PI3Kα Inhibition in <i>PIK3CA</i> Mutant Breast Epithelial Cells

Activating PIK3CA mutations are among the most frequent oncogenic drivers in breast cancer, with E545K and H1047R mutants representing the most prevalent hotspot variants. Despite the development of potent PI3K inhibitors, clinical efficacy remains limited in some cases. This underscores the need to understand how specific oncogenic PIK3CA mutations reshape signaling networks and therapeutic responses. Here, we compared the E545K and H1047R mutant breast epithelial cells to delineate mutation-specific signaling programs, growth phenotypes, and responses to PI3Kα inhibition in the presence and absence of insulin, using integrated growth assays and quantitative proteomic and phosphoproteomic profiling. These analyses uncovered mutation-specific signaling architectures and inhibitor sensitivities. Both mutants exhibited basal MAPK activation, but showed divergent MAPK phosphorylation dynamics in distinct PIK3CA mutations, suggesting a pivotal role for MAPK signaling. Upon PI3Kα inhibition with alpelisib, insulin engaged bypass signaling that partially counteracted downstream suppression. MEK inhibition alone suppressed the growth of PIK3CA mutant cells, and dual targeting of PI3K and MAPK signaling produced greater growth suppression than either single agent alone under insulin-stimulated conditions. Collectively, these findings reveal mutation-specific adaptive signaling and support combined PI3Kα and MAPK pathway inhibition as a strategy to improve therapeutic efficacy in PIK3CA mutant breast cancer.