The CBL Knockout Huh-7 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from the Huh-7 human hepatocellular carcinoma line, with targeted disruption of the CBL gene. This loss-of-function model is provided as a heterogeneous polyclonal pool, avoiding clonal artifacts and reflecting the genetic diversity of tumor cell populations. CRISPR/Cas9-mediated gene disruption abolishes CBL E3 ubiquitin ligase activity, impairing its role in receptor tyrosine kinase (RTK) downregulation. The knockout cells lack functional CBL protein, enabling investigation of sustained RTK signaling and associated phenotypic changes.
The parental Huh-7 cell line was established from a well-differentiated hepatocellular carcinoma of a Japanese male and is a widely used model for liver cancer research. These adherent epithelial cells retain key signaling pathways relevant to hepatocarcinogenesis, including MET, EGFR, and PDGFR axes. Their well-characterized biology and genetic tractability make them suitable for dissecting the role of tumor suppressors and oncogenes in hepatocellular carcinoma progression. Huh-7 cells are frequently employed in studies of drug sensitivity, migration, and signal transduction, providing a robust platform for functional genomics.
CBL encodes an E3 ubiquitin ligase that negatively regulates RTK signaling. Upon ligand binding (e.g., EGF, HGF, PDGF) to EGFR, MET, or PDGFR, CBL is recruited via adaptors GRB2 and CIN85. It ubiquitinates activated receptors, promoting their clathrin-mediated endocytosis through interaction with endophilin, and targeting them for lysosomal or proteasomal degradation. CBL also interacts with SRC family kinases and modulates signaling by SYK, ZAP70, and PI3K. In knockout cells, loss of CBL disrupts this negative feedback, leading to sustained surface expression of EGFR, MET, and PDGFR and persistent activation of downstream effector pathways.
In hepatocellular carcinoma, CBL acts as a tumor suppressor, and its loss is associated with enhanced proliferation and invasion. The Huh-7 CBL knockout model allows functional studies of CBL deficiency in a liver cancer background. Without CBL-mediated downregulation, MET signaling driven by HGF can become constitutively active, promoting epithelial-mesenchymal transition, cell migration, and invasion. Sustained EGFR and PDGFR signals further enhance tumorigenic properties. This polyclonal population is particularly useful for examining the interplay between CBL loss and oncogenic RTK networks in hepatocarcinogenesis.
Key applications include quantitative analysis of RTK turnover using cycloheximide chase assays and immunoblotting for phospho-EGFR/MET; flow cytometry to measure surface receptor levels; and Transwell migration assays to evaluate HGF-induced invasion. The cells support phospho-RTK arrays, co-immunoprecipitation of interacting partners, and RT-qPCR for downstream targets. They are also amenable to synthetic lethal screens and testing resistance to MET or EGFR inhibitors. For additional details or custom inquiries, please contact Ascent Research.
