Functional characterization of PIK3CA E545A mutation in MCF-7 breast cancer cells reveals enhanced proliferation and resistance to Alpelisib

PIK3CA mutations are central oncogenic drivers in hormone receptor-positive, HER2-negative breast cancer; however, the functional and therapeutic relevance of noncanonical variants remains incompletely defined. The E545A mutation, increasingly reported in specific patient populations, has not been systematically investigated. We generated an isogenic MCF-7 cell model harboring the PIK3CA E545A mutation using CRISPR/Cas9-mediated homology-directed repair to delineate its phenotypic and pharmacological consequences. E545A induced a robust gain-of-function phenotype, characterized by a mesenchymal-like morphological transition with reduced circularity and decreased cell size. This structural shift was accompanied by enhanced tumor cell fitness, including accelerated proliferation kinetics, increased metabolic activity, and significantly elevated clonogenic capacity compared with wild-type controls. Notably, growth trajectories showed sustained divergence between mutant and control cells across all time points, indicating a stable proliferative advantage. Importantly, E545A conferred diminished sensitivity to the PI3Kα Inhibitor Alpelisib. Mutant cells retained migratory capacity under treatment and exhibited a pronounced, time-dependent increase in IC₅₀, consistent with adaptive resistance. Collectively, these findings identify E545A as a functionally active and therapeutically consequential PIK3CA variant. Our study expands the current understanding of PIK3CA-driven oncogenic diversity beyond canonical hotspot mutations and underscores the need for variant-resolved stratification to improve the efficacy of PI3K-targeted therapies.