Tian Yu, Bao Xin, Lei Shan, Huang Youcai, Wang Xiaoling, Tu Yanyang, He Qinglian, Zhang Feixiang, Xu Haicheng, Ashrafizadeh Milad, Sethi Gautam, Wang Furong, Zeng Zhirui
1Science Research Center,the Huizhou Central People's Hospital, Guangdong Medical University, Huizhou, Guangdong, China.
Engineering Research Center of Chronic Disease Diagnosis and Treatment, School of Basic Medicine, Guizhou Medical University, No. 9 Beijing Road, Guiyang, Guizhou, 550009, China.
Cell Commun Signal. 2025 Jul 10;23(1):329. doi: 10.1186/s12964-025-02285-x.
Sorafenib, a ferroptosis agonist, is a first-line treatment for advanced hepatocellular carcinoma (HCC). However, its clinical efficacy is limited due to drug resistance, resulting in modest improvements in patient survival. Hence, the present study has been designed to identify critical molecular targets associated with sorafenib resistance and investigate the potential inhibitors in overcoming this therapeutic challenge.
In vivo whole-genome CRISPR/Cas9 library screens were conducted to identify resistance factors to ferroptosis agonists, such as RSL3 and sorafenib, in HCC. The effects and underlying molecular mechanisms of these resistance factors were investigated in HCC cells using ferroptosis detection assays, xenograft tumor models, chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assays. Potential inhibitors targeting these factors were evaluated through computer-aided virtual screening, molecular dynamics simulations, surface plasmon resonance analysis, and functional evaluations.
A retinoic acid metabolism gene cluster, including ADH4, ALDH1A1, ALDH1A3, FABP5, RBP1, and RDH10, was found demonstrating upregulation in HCC cells treated with ferroptosis agonist, sorafenib. This gene cluster contributes to the ferroptosis resistance by producing the strong reducing agent retinoic acid. The transcription factor POU3F3 was identified as a key regulator for the retinoic acid metabolism gene cluster, which simultaneously binds to their promoters, increasing their transcription and promoting retinoic acid production. Knockdown of POU3F3 significantly enhanced the pro-ferroptotic and inhibitory effects of sorafenib on HCC cells by suppressing retinoic acid metabolism. Furthermore, rosarin was identified as a POU3F3 inhibitor, with an equilibrium dissociation constant of 7.57 µM, and demonstrated a synergistic effect with sorafenib against HCC cells both in vitro and in vivo.
According to the results, POU3F3 acts as a protective regulator against sorafenib-induced ferroptosis in HCC cells by enhancing the transcription of multiple retinoic acid metabolism genes and promoting retinoic acid production. The POU3F3 inhibitor, rosarin, shows potential as an ideal candidate for overcoming sorafenib resistance in HCC.
索拉非尼是一种铁死亡激动剂，是晚期肝细胞癌（HCC）的一线治疗药物。然而，由于耐药性，其临床疗效有限，患者生存率的改善不大。因此，本研究旨在确定与索拉非尼耐药相关的关键分子靶点，并研究克服这一治疗挑战的潜在抑制剂。
进行体内全基因组CRISPR/Cas9文库筛选，以确定肝癌细胞中对铁死亡激动剂（如RSL3和索拉非尼）的耐药因子。使用铁死亡检测试验、异种移植肿瘤模型、染色质免疫沉淀（ChIP）和双荧光素酶报告试验，在肝癌细胞中研究这些耐药因子的作用及其潜在分子机制。通过计算机辅助虚拟筛选、分子动力学模拟、表面等离子体共振分析和功能评估，评估靶向这些因子的潜在抑制剂。
发现一个视黄酸代谢基因簇，包括ADH4、ALDH1A1、ALDH1A3、FABP5、RBP1和RDH10，在用铁死亡激动剂索拉非尼处理的肝癌细胞中呈上调。该基因簇通过产生强还原剂视黄酸来促进铁死亡抗性。转录因子POU3F3被确定为视黄酸代谢基因簇的关键调节因子，它同时与它们的启动子结合，增加它们的转录并促进视黄酸的产生。敲低POU3F3通过抑制视黄酸代谢，显著增强了索拉非尼对肝癌细胞的促铁死亡和抑制作用。此外，玫瑰树碱被鉴定为一种POU3F3抑制剂，平衡解离常数为7.57 μM，并在体外和体内均显示出与索拉非尼对肝癌细胞的协同作用。
结果表明，POU3F3通过增强多个视黄酸代谢基因的转录并促进视黄酸的产生，作为肝癌细胞中索拉非尼诱导的铁死亡的保护调节因子。POU3F3抑制剂玫瑰树碱显示出作为克服肝癌中索拉非尼耐药的理想候选药物的潜力。
