Macropinocytosis is an alternative pathway of cysteine acquisition and mitigates sorafenib-induced ferroptosis in hepatocellular carcinoma

Background: Macropinocytosis, an important nutrient-scavenging pathway in certain cancer cells, allows cells to compensate for intracellular amino acid deficiency under nutrient-poor conditions. Ferroptosis caused by cysteine depletion plays a pivotal role in sorafenib responses during hepatocellular carcinoma (HCC) therapy. However, it is not known whether macropinocytosis functions as an alternative pathway to acquire cysteine in sorafenib-treated HCC, and whether it subsequently mitigates sorafenib-induced ferroptosis. This study aimed to investigate whether sorafenib drives macropinocytosis induction, and how macropinocytosis confers ferroptosis resistance on HCC cells. Methods: Macropinocytosis, both in HCC cells and HCC tissues, was evaluated by measuring TMR-dextran uptake or lysosomal degradation of DQ-BSA, and ferroptosis was evaluated via C11-BODIPY fluorescence and 4-HNE staining. Sorafenib-induced ferroptosis and macropinocytosis were validated in tumor tissues taken from HCC patients who underwent ultrasound-guided needle biopsy. Results: Sorafenib increased macropinocytosis in human HCC specimens and xenografted HCC tissues. Sorafenib-induced mitochondrial dysfunction was responsible for activation of PI3K-RAC1-PAK1 signaling, and amplified macropinocytosis in HCC. Importantly, macropinocytosis prevented sorafenib-induced ferroptosis by replenishing intracellular cysteine that was depleted by sorafenib treatment; this rendered HCC cells resistant to sorafenib. Finally, inhibition of macropinocytosis by amiloride markedly enhanced the anti-tumor effect of sorafenib, and sensitized resistant tumors to sorafenib. Conclusion: In summary, sorafenib induced macropinocytosis, which conferred drug resistance by mitigating sorafenib-induced ferroptosis. Thus, targeting macropinocytosis is a promising therapeutic strategy to facilitate ferroptosis-based therapy for HCC. Graphic Abstract: [Figure not available: see fulltext.]