A genome-wide screen identified ARHGAP35 as a regulator of regorafenib resistance in liver cancer.

Regorafenib, a multikinase inhibitor, is widely used to treat hepatocellular carcinoma. However, chemoresistance poses a significant challenge to its long-term efficacy. This study conducted a genome-wide CRISPR/Cas9 knockout screen in liver cancer cell lines to identify key regulators of regorafenib resistance and elucidate the underlying molecular mechanisms. The screen identified ARHGAP35 as a critical negative regulator of regorafenib resistance. ARHGAP35 depletion conferred resistance in HepG2 and Huh7 cells, while regorafenib-resistant variants (HepG2-R and Huh7-R) exhibited decreased ARHGAP35 expression. Reintroducing ARHGAP35 restored drug sensitivity. Further analysis revealed that reduced ARHGAP35 expression facilitated epithelial-mesenchymal transition (EMT) by activating the RhoA signaling pathway. Notably, RhoA inhibition reversed EMT and restored regorafenib sensitivity. These findings highlight ARHGAP35 as a key modulator of regorafenib resistance through RhoA suppression, offering potential therapeutic targets to combat chemoresistance in liver cancer.