The ITCH Knockout HCT 116 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from the human HCT 116 colorectal carcinoma cell line, in which the ITCH gene has been disrupted to eliminate functional ITCH expression. This polyclonal population, generated without single-cell cloning, retains the heterogeneous genetic background of the parental line while introducing loss-of-function mutations across the ITCH locus, enabling robust studies of ITCH-dependent pathways without the confounding effects of clonal selection.
The HCT 116 host cell line is a near-diploid, adherent human colorectal carcinoma model characterized by a KRAS G13D activating mutation and MLH1 deficiency, resulting in microsatellite instability (MSI-H). Originating from malignant colonic epithelial cells, HCT 116 cells are widely used in cancer research to dissect oncogenic signaling, DNA mismatch repair, and tumor suppressor pathways, making them a relevant system for investigating the role of E3 ubiquitin ligases in colorectal tumorigenesis.
ITCH encodes a HECT-type E3 ubiquitin ligase that covalently attaches ubiquitin to substrate proteins, targeting them for proteasomal degradation. ITCH is activated by phosphorylation through upstream kinases such as JNK (MAPK8/9) and AKT, and is regulated by interactions with NDFIP1 and UbcH7. It mediates degradation of substrates including c-FLIP (CFLAR), JunB, p73 (TP73), p63 (TP63), LATS1, BCL10, NOTCH1, SOCS1, and Smad2/3, thereby controlling apoptosis, immune signaling, and cell proliferation. Specifically, ITCH suppresses pro-survival signals by degrading c-FLIP and modulates Hippo pathway activity by targeting LATS1 for destruction, which in turn affects YAP1 stability. In addition, ITCH participates in TNF-alpha-induced NF-kappaB activation and T cell receptor signaling, highlighting its multifaceted role in cellular homeostasis and inflammation.
In the HCT 116 colorectal cancer background, loss of ITCH E3 ligase activity abrogates ubiquitination of its substrates, leading to accumulation of proteins such as JunB and p73, which can alter the balance between apoptosis and proliferation. This is particularly significant given the KRAS G13D mutation, which drives constitutive MAPK pathway activity, and the MSI-H status, which influences genomic stability and immune responses. The ITCH knockout model in this line thus provides a powerful tool to study the intersection of ubiquitin-mediated proteolysis with MAPK, NF-kB, JNK, and Hippo signaling cascades in a malignancy-relevant context. It also enables investigation of how ITCH deficiency may exacerbate inflammatory phenotypes associated with inflammatory bowel disease and colorectal cancer progression.
Researchers can employ this ITCH knockout polyclonal cell population for a broad range of functional assays, including Western blotting to assess substrate accumulation, ubiquitination assays to monitor ligase activity, and co-immunoprecipitation to validate ITCH-substrate interactions. The model is well-suited for apoptosis studies using Annexin V/PI staining, cell viability and migration assays, and flow cytometry-based analyses of signaling pathway activation. Additionally, RT-qPCR and immunofluorescence can be used to track transcriptional changes and subcellular localization of ITCH targets. Applications include mechanistic dissection of E3 ligase function, validation of drug candidates targeting the ubiquitin-proteasome system, and exploration of crosstalk between Hippo, Wnt, and TGF-beta pathways in colorectal cancer. For further information or technical support related to this knockout model, please contact Ascent Research.
