CUL1-neddylation contributes to K29-linked ubiquitination on p27 for autophagic degradation in sorafenib-resistant liver cancer.

Sorafenib has demonstrated great efficacy in liver cancer, however, its application as first-line treatment has been hampered due to the emerging drug resistance. This study is aimed to investigate the mechanism underlying acquired sorafenib resistance in liver cancer. Based on GSE109211 and TCGA datasets, bioinformatics analysis was conducted to find the potential genes implicated in the sorafenib resistance in liver cancer. mCherry-/eGFP-LC3B dual-fluorescent system was used to assess autophagic state. Wild and mutant types of HA-labeled ubiquitin (K27, K29, K33, K48, K63, K29R and K48R) were used to identify the type of polyubiquitin chains added to p27 by CUL1. Herein, we identified that F-box protein (SCF) ubiquitin ligase complexes (CUL1 and SKP2) and NEDD8 were highly expressed in sorafenib-resistant tissues using both the public data and clinical samples. NEDD8-mediated CUL1 neddylation enhanced SCF ubiquitin ligase complex to target p27 and subsequently linked K29-linked polyubiquitin chains to p27. Furthermore, NBR1 facilitated the degradation of ubiquitinated p27 protein by enhancing autophagy flux. Knocking down of CUL1 could prevent ubiquitination- and autophagy-mediated p27 protein degradation. The resistance to sorafenib was suppressed with CUL1 knockdown both in vitro and in vivo. In conclusion, our findings indicated that blocking neddylation or autophagy can restore drug sensitivity, thus providing a potential strategy for overcoming sorafenib resistance in the future.