CCT3/ACTN4/TFRC axis protects hepatocellular carcinoma cells from ferroptosis by inhibiting iron endocytosis

Sorafenib is widely used in treating advanced hepatocellular carcinoma (HCC). However, its effectiveness in prolonging patient survival is limited by the development of drug resistance. To systematically investigate the resistance mechanisms of Sorafenib, an integrative analysis combining posttranslational modification (PTM) omics and CRISPR/Cas9 knockout library screening was conducted. This analysis identified ubiquitination at lysine 21 (K21) on chaperonin-containing TCP1 subunit 3 (CCT3) as being associated with Sorafenib resistance. Transcriptomic data from HCC patients treated with Sorafenib revealed that CCT3 expression was lower in responders compared to non-responders. Experimentally, inhibiting the expression of CCT3 sensitized HCC cells to Sorafenib and enhanced Sorafenib-induced ferroptosis. Additionally, CCT3 was found to interact with ACTN4, hindering the recycling of transferrin receptor protein 1 (TFRC) to the cell membrane, thus obstructing iron endocytosis. Mechanistically, the inhibition of ferroptosis by CCT3 depends on the deubiquitination of K6-linked non-degradative ubiquitination at its K21, which occurs upon Sorafenib treatment. Moreover, CCT3 knockdown enhanced the anti-tumor effects of Sorafenib in nude mice. In summary, we have identified a novel function of the chaperone protein. Targeting the CCT3/ACTN4/TFRC axis offers a promising strategy to enhance ferroptosis and overcome Sorafenib resistance in HCC.