NHSL3 knockout modulates sorafenib response, growth, and motility in hepatocellular carcinoma cell models.

backgroundNance-Horan syndrome-like protein 3 (NHSL3) has been implicated in hepatocellular carcinoma (HCC) progression, but its role in sorafenib response and related growth and motility phenotypes remains unclear. In this study, we examined NHSL3 loss in Huh-7 cells and selected phenotypes in Hep3B cells. METHODS AND

resultsNHSL3 knockout clones were generated in both cell lines by paired Cas9 nickase editing. Data were obtained from three independent biological experiments. In Huh-7 cells, NHSL3 loss increased sorafenib sensitivity, lowering the 24 h half-maximal inhibitory concentration from 13.4 µM to 5.8 µM. NHSL3 loss was associated with G1 enrichment, reduced S-phase fraction, and impaired clonogenic growth. It also delayed wound closure and reduced transwell migration, whereas invasion varied with sorafenib exposure and assay duration. In Hep3B cells, the genotype-dependent sorafenib response was more modest and emerged at 48 h. NHSL3 loss nevertheless reproduced the reduced clonogenic growth phenotype and was associated with lower migration and invasion under vehicle-treated conditions. Molecular analyses in Huh-7 cells showed selective epithelial-mesenchymal transition (EMT)-associated remodeling, including a blunted E-Cadherin response to sorafenib and increased Vimentin and N-Cadherin levels. NHSL3 loss was also associated with lower basal phosphorylated RAF1 levels and confluence- and sorafenib-dependent YAP/TAZ responses.

conclusionsNHSL3 loss was associated with reduced clonogenic growth and migration across two HCC cell backgrounds, whereas sorafenib response differed between models. These findings extend the functional scope of NHSL3 to sorafenib response in HCC. In Huh-7 cells, these changes coincided with EMT-associated protein remodeling and growth-related signaling alterations.