肝细胞癌（HCC）是全球最常见和致命的癌症之一，但目前的治疗选择仍然有限并且会导致严重的危及生命的副作用。异常的FGFR4信号被验证为HCC的致癌驱动因子，而EZH2，PRC2复合物的催化亚单位，是许多肿瘤，包括HCC获得药物耐药性的潜在因素。然而，这两个致癌因子之间的功能关系，特别是它们对HCC治疗的重要性仍不清楚。在本研究中，我们系统评估了针对FGFR4和EZH2的联合治疗对HCC的可行性。分析了The Cancer Genome Atlas（TCGA）中肝细胞癌（LIHC）患者的RNA测序数据，以确定FGFR4和EZH2的表达及其与预后的相互作用。此外，使用FGFR4抑制剂（Roblitinib）和/或EZH2抑制剂（CPI-169）处理了HCC细胞系、斑马鱼/小鼠HCC异位移植瘤和斑马鱼HCC原发性肿瘤，然后进行了细胞增殖、存活、凋亡和肿瘤生长分析，以评估联合治疗在HCC体内外的可行性。此外，RNA-Seq与ChIP-Seq数据分析相结合，研究了Roblitinib和CPI-169联合治疗的关键机制。EZH2在响应FGFR4抑制剂治疗时通过非规范的NF-kB信号积累，提高的EZH2水平导致HCC对Roblitinib（FGFR4抑制剂）的拮抗作用。值得注意的是，EZH2的沉默增强了HCC细胞对Roblitinib的敏感性，而Roblitinib和CPI-169（EZH2抑制剂）的联合治疗在体外协同诱导了HCC细胞凋亡，并抑制了在体内斑马鱼/小鼠HCC异位移植瘤和斑马鱼HCC原发性肿瘤的发展。此外，Roblitinib和CPI-169通过抑制YAP信号通路协同抑制了HCC的发展。总之，本研究突显了FGFR4和EZH2抑制剂联合治疗的潜力，这将为HCC的临床治疗策略的进一步发展提供新的参考。©2023.作者及版权所有。

Hepatocellular carcinoma (HCC) is one of the most common and lethal cancers worldwide, but current treatment options remain limited and cause serious life-threatening side effects. Aberrant FGFR4 signaling has been validated as an oncogenic driver of HCC, and EZH2, the catalytic subunit of the PRC2 complex, is a potential factor that contributes to acquired drug resistance in many tumors, including HCC. However, the functional relationship between these two carcinogenic factors, especially their significance for HCC treatment, remains unclear. In this study, we systematically evaluated the feasibility of a combination therapy targeting FGFR4 and EZH2 for HCC.RNA sequencing data of patients with Liver hepatocellular carcinoma (LIHC) from The Cancer Genome Atlas (TCGA) were analyzed to determine FGFR4 and EZH2 expression and their interaction with prognosis. Moreover, the HCC cell lines, zebrafish/mouse HCC xenografts and zebrafish HCC primary tumors were treated with FGFR4 inhibitor (Roblitinib) and/or EZH2 inhibitor (CPI-169) and then subjected to cell proliferation, viability, apoptosis, and tumor growth analyses to evaluate the feasibility of combination therapy for HCC both in vitro and in vivo. Furthermore, RNA-Seq was performed in combination with ChIP-Seq data analysis to investigate the critical mechanism underlying the combination treatment with Roblitinib and CPI-169.EZH2 accumulated through the non-canonical NF-kB signaling in response to FGFR4 inhibitor treatment, and the elevated EZH2 levels led to the antagonism of HCC against Roblitinib (FGFR4 inhibitor). Notably, knockdown of EZH2 sensitized HCC cells to Roblitinib, while the combination treatment of Roblitinib and CPI-169 (EZH2 inhibitor) synergistically induced the HCC cell apoptosis in vitro and suppressed the zebrafish/mouse HCC xenografts and zebrafish HCC primary tumors development in vivo. Moreover, Roblitinib and CPI-169 synergistically inhibited HCC development via repressing YAP signaling.Collectively, our study highlighted the potential of the therapeutic combination of FGFR4 and EZH2 inhibitors, which would provide new references for the further development of clinical treatment strategies for HCC.© 2023. The Author(s).