C/EBP-β Mediates the Reversal of Sorafenib Resistance by Tunicamycin in Hepatocellular Carcinoma

Introduction: The multikinase inhibitor sorafenib is a standard-of-care therapy for advanced hepatocellular carcinoma (HCC). However, acquired resistance to sorafenib severely compromises its long-term clinical efficacy. This study aimed to investigate whether the endoplasmic reticulum (ER) stress inducer tunicamycin (TM) could overcome this resistance and to elucidate the central role of the transcription factor CCAAT/enhancer-binding protein β (C/EBP-β) in this process. Methods: We established sorafenib-resistant hepatocellular carcinoma HepG2 and Huh7 cell lines to mimic the resistant phenotype. The sensitizing effect of TM was evaluated by assessing proliferation, colony formation, reactive oxygen species (ROS) production, and apoptosis. Therapeutic efficacy was further validated in vivo using subcutaneous and orthotopic xenograft models. Results: We found that TM significantly inhibited the proliferation and clonogenicity of sorafenib-resistant cells. TM treatment upregulated C/EBP-β expression and restored sensitivity to sorafenib. Mechanistically, C/EBP-β overexpression phenocopied the effect of TM, whereas its knockdown attenuated TM-induced sensitization. Furthermore, TM induced a C/EBP-β-dependent increase in intracellular ROS, triggering the intrinsic apoptotic pathway characterized by p53 accumulation, an increased Bax/Bcl-2 ratio, and caspase-3 cleavage. In vivo, the combination of TM and C/EBP-β overexpression exerted potent antitumor effects, reducing tumor volume by approximately 91% relative to the sorafenib-only control group in subcutaneous models. Conclusion: Our study identified C/EBP-β as a key effector of the terminal pro-apoptotic ER stress response. While the direct clinical application of TM is limited by its systemic toxicity, these findings highlight the clinical translational potential of targeting the ER stress-C/EBP-β axis, offering a novel combination therapeutic strategy for patients with acquired sorafenib resistance. Keywords: hepatocellular carcinoma, HCC, sorafenib-resistance, endoplasmic reticulum, ER stress, tunicamycin, C/EBP-β, reactive oxygen species, ROS