E3 ubiquitin-ligase Hakai regulates LRP4 stability and Wnt/β-catenin signalling in colorectal cancer cells.

The epithelial-mesenchymal transition is associated with the acquisition of cancer stem cell (CSC) traits that promote metastasis and therapy resistance. Here, we investigated the role of the E3 ubiquitin-ligase Hakai in regulating CSC-associated properties in colorectal cancer. Using tumoursphere models, loss-of-function approaches, proteomics, bioinformatics, and Wnt/β-catenin pathway studies, we found that Hakai depletion reduced tumoursphere formation, CSC marker expression, and Wnt target gene activation. Mechanistically, our findings support that Hakai regulates LRP4 stability, a negative regulator of Wnt signaling, consistent with ubiquitin-dependent mechanism. Consequently, Hakai enhances β-catenin/TCF transcriptional activity, nuclear accumulation of β-catenin, and attenuation of LRP4-mediated inhibition of Wnt signaling. Pharmacological inhibition of Hakai with Hakin-1, a selective inhibitor of its HYB domain responsible for E3 ubiquitin-ligase activity, reduced tumoursphere formation and promoted differentiation-associated features. Furthermore, these findings highlight Hakai inhibition as a potential CSC-directed therapeutic strategy in colorectal cancer.