The EFNA5 Knockout HCT 116 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout pool derived from the HCT 116 colorectal carcinoma line, featuring disruption of the EFNA5 gene. Ephrin-A5, the encoded GPI-anchored ligand, mediates EphA receptor bidirectional signaling that regulates cell adhesion and repulsion. The heterogeneous knockout population provides a loss-of-function model for studying adhesion, migration, and cancer biology without clonal selection artifacts.
HCT 116 is a human colorectal adenocarcinoma cell line with microsatellite instability (MSI-H), a near-diploid karyotype, and well-characterized oncogenic mutations in KRAS (G13D) and PIK3CA (H1047R). These alterations drive constitutive activation of the MAPK/ERK and PI3K/AKT pathways, establishing this line as a robust model for investigating colorectal cancer progression and metastasis in an epithelial context.
Ephrin-A5 binds EphA receptors (notably EphA3, EphA4, EphA5) to trigger bidirectional signaling: forward signaling activates Src family kinases and FAK, while reverse signaling recruits adaptors Grb4 and Nck. These events modulate Rho GTPases (RhoA, Rac1, Cdc42), controlling actin dynamics and integrin-based adhesion. EFNA5 expression is regulated by p53, Wnt/??-catenin, and DNA methylation, and its signaling intersects with EGFR and MAPK/ERK and PI3K/AKT pathways. Proteolytic shedding by ADAM10 further tunes ephrin-A5 activity, positioning it at a nexus of cell adhesion and survival networks.
In the HCT 116 background, where oncogenic KRAS and PIK3CA mutations drive constitutive MAPK and PI3K/AKT signaling, EFNA5 knockout is expected to impair Eph receptor-mediated regulation of the actin cytoskeleton via Rho GTPases. This may attenuate cell migration and invasion, which are critical for metastasis. Additionally, altered crosstalk with EGFR and ADAM10-mediated shedding could influence tumor microenvironment interactions. This provides a valuable system for deciphering ephrin-A5 function in a clinically relevant mutational context.
These knockout cells enable in-depth investigation of colorectal cancer metastasis and Eph/ephrin signaling. Applications include transwell migration and invasion assays to assess metastatic capability, wound healing assays for directional movement, and immunofluorescence microscopy to examine actin cytoskeleton and focal adhesion reorganization. Biochemical analyses such as western blotting for EphA4, phospho-ERK1/2, and phosphorylated AKT probe signaling consequences, while co-immunoprecipitation identifies changes in Eph receptor complexes. RNA sequencing provides transcriptome-wide profiling of ephrin-A5-dependent pathways, and apoptosis assays evaluate cell survival. For in vivo studies, the cells are suitable for xenograft tumorigenesis models. For further technical details, please contact Ascent Research.
