Cell J. 2025 Dec 22. pii: 731598. [Epub ahead of print]27(1):
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Objective: The PI3K/Akt signaling pathway plays a central role in regulating cell growth, survival, and metabolism, and its dysregulation is a hallmark of many cancers. The PIK3CA gene, which encodes the alpha catalytic subunit of PI3K, is altered in approximately 30% of breast cancers. Among its mutations, c.3140A>G (p.His1047Arg) in the kinase domain is the most prevalent, producing a constitutively active enzyme with oncogenic potential. Here, we engineered a population of MCF7 cells carrying the PIK3CA c.3140A>G mutation using CRISPR-Cas9 with precise single-nucleotide editing, and evaluated its impact on cellular characteristics.
Materials and Methods: In this experimental study, nearly homogeneous populations of PIK3CA H1047R mutant MCF7 cells were generated using CRISPR-Cas9-mediated genome editing followed by hierarchical single-cell isolation. Editing efficiency was validated through allele-specific polymerase chain reaction (PCR) and multiple rounds of Sanger sequencing. Cell cycle distribution and proliferation were analyzed using flow cytometry and cell count assays, respectively. Gene expression changes were assessed by quantitative real-time PCR to evaluate the mutation's impact on cell cycle-related genes.
Results: Tracking of insertions, deletions, and recombination events (TIDER) analysis showed approximately 60% homology-directed repair (HDR) efficiency in the edited population. Flow cytometry revealed a 5% increase in the G2/M cell population in the edited clone compared with unedited controls (P<0.001). Proliferation assays demonstrated significantly accelerated growth (1.30 fold) under low fetal bovine serum (FBS) conditions (P=0.029). Quantitative real-time PCR confirmed upregulation of cell cycle-promoting genes, with CCND1 and MYC expression increasing by 1.62-fold (P<0.001) and 1.23-fold (P<0.001), respectively, relative to controls.
Conclusion: The genetically edited cell lines represent robust and well-defined experimental models that enable direct assessment of the functional consequences of oncogenic driver mutations on cellular behavior and signaling pathways. Our findings demonstrate that targeted genetic alterations induce measurable changes in proliferation, cell-cycle regulation, and gene expression, thereby providing mechanistic insight into tumorigenesis and the specific contribution of driver mutations to cancer-related cellular phenotypes.
Keywords: Breast Cancer; CRISPR-Cas9; Genome EditingGenome Editing; MCF7 Cell Line; PIK3CA