The ATP1A3 Knockout HCT 116 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal population derived from the HCT 116 human colorectal carcinoma cell line, designed to disrupt ATP1A3 gene expression. This mixed knockout pool minimizes clonal artifacts and provides a robust loss-of-function model for studying Na+/K+-ATPase alpha-3 subunit deficiency in an epithelial cancer context.
HCT 116 is an established colorectal carcinoma model with epithelial morphology and near-diploid karyotype. It harbors KRAS (G13D) and PIK3CA mutations, which constitutively activate MAPK/ERK and PI3K/AKT oncogenic signaling. These features make the cell line highly relevant for investigating pathway crosstalk, drug responses, and tumor biology mechanisms central to colorectal cancer research.
ATP1A3 encodes the alpha-3 isoform of the Na+/K+-ATPase, a plasma membrane pump critical for maintaining Na+ and K+ gradients, cell volume, and membrane potential. Beyond ion transport, the pump functions as a signaling scaffold that interacts with Src kinase, caveolin-1, ankyrin, and 14-3-3 proteins. Cardiac glycosides (ouabain, digoxin) bind ATP1A3 and modulate Src activation, leading to EGFR transactivation and stimulation of downstream MAPK/ERK (HRAS, MAP2K1, MAPK1) and PI3K/AKT (PIK3CA, AKT1) cascades. This signaling also influences intracellular Ca2+ via NCX1 and ITPR1, and transcriptionally regulates cell cycle factors like cyclin D1 and p21. Beta subunits (ATP1B) and FXYD proteins further modulate pump activity, while PKA and PKC provide regulatory phosphorylation.
ATP1A3 disruption in HCT 116 cells likely disturbs ion homeostasis, altering the Src-mediated signaling network that intersects with the line??s oncogenic driver mutations. Consequently, MAPK/ERK and PI3K/AKT pathway activities may be imbalanced, affecting proliferation, migration, apoptosis, and drug sensitivity. This knockout model enables dissection of the interplay between Na+/K+-ATPase function and colorectal cancer signaling, potentially identifying therapeutic targets or resistance mechanisms linked to ATP1A3.
Recommended applications include Western blotting and RT-qPCR for confirming ATP1A3 loss, and functional assays such as MTT/BrdU proliferation, Transwell migration/invasion, and flow cytometry for cell cycle/apoptosis analysis. The model is suitable for evaluating cardiac glycoside and Src inhibitor responses, measuring intracellular Na+/K+ levels, and profiling phospho-signaling events (phospho-Src, phospho-ERK) by immunoblotting or array. RNA-seq provides transcriptome-wide insights. For further technical details, validation data, or ordering inquiries, please contact Ascent Research.
