Department of Gastroenterology, Institute of Liver and Gastrointestinal Diseases, Hubei Key Laboratory of Hepato-Pancreato-Biliary Diseases, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Department of Gastroenterology, Shangrao People's Hospital, Shangrao, China.
J Hepatol. 2023 Jul;79(1):109-125. doi: 10.1016/j.jhep.2023.02.036. Epub 2023 Mar 11.
BACKGROUND & AIMS: Metastasis remains the major reason for the high mortality of patients with hepatocellular carcinoma (HCC). This study was designed to investigate the role of E-twenty-six-specific sequence variant 4 (ETV4) in promoting HCC metastasis and to explore a new combination therapy strategy for ETV4-mediated HCC metastasis. METHODS: PLC/PRF/5, MHCC97H, Hepa1-6, and H22 cells were used to establish orthotopic HCC models. Clodronate liposomes were used to clear macrophages in C57BL/6 mice. Gr-1 monoclonal antibody was used to clear myeloid-derived suppressor cells (MDSCs) in C57BL/6 mice. Flow cytometry and immunofluorescence were used to detect the changes of key immune cells in the tumour microenvironment. RESULTS: ETV4 expression was positively related to higher tumour-node-metastasis (TNM) stage, poor tumour differentiation, microvascular invasion, and poor prognosis in human HCC. Overexpression of ETV4 in HCC cells transactivated PD-L1 and CCL2 expression, which increased tumour-associated macrophage (TAM) and MDSC infiltration and inhibited CD8 T-cell accumulation. Knockdown of CCL2 by lentivirus or CCR2 inhibitor CCX872 treatment impaired ETV4-induced TAM and MDSC infiltration and HCC metastasis. Furthermore, FGF19/FGFR4 and HGF/c-MET jointly upregulated ETV4 expression through the ERK1/2 pathway. Additionally, ETV4 upregulated FGFR4 expression, and downregulation of FGFR4 decreased ETV4-enhanced HCC metastasis, which created a FGF19-ETV4-FGFR4 positive feedback loop. Finally, anti-PD-L1 combined with FGFR4 inhibitor BLU-554 or MAPK inhibitor trametinib prominently inhibited FGF19-ETV4 signalling-induced HCC metastasis. CONCLUSIONS: ETV4 is a prognostic biomarker, and anti-PD-L1 combined with FGFR4 inhibitor BLU-554 or MAPK inhibitor trametinib may be effective strategies to inhibit HCC metastasis. IMPACT AND IMPLICATIONS: Here, we reported that ETV4 increased PD-L1 and chemokine CCL2 expression in HCC cells, which resulted in TAM and MDSC accumulation and CD8 T-cell inhibition to facilitate HCC metastasis. More importantly, we found that anti-PD-L1 combined with FGFR4 inhibitor BLU-554 or MAPK inhibitor trametinib markedly inhibited FGF19-ETV4 signalling-mediated HCC metastasis. This preclinical study will provide a theoretical basis for the development of new combination immunotherapy strategies for patients with HCC.
背景与目的：转移仍然是导致肝细胞癌（HCC）患者高死亡率的主要原因。本研究旨在探讨 E-二十六位特异性序列变异体 4（ETV4）在促进 HCC 转移中的作用，并探索一种新的 ETV4 介导的 HCC 转移的联合治疗策略。 方法：使用 PLC/PRF/5、MHCC97H、Hepa1-6 和 H22 细胞建立原位 HCC 模型。使用氯膦酸盐脂质体清除 C57BL/6 小鼠中的巨噬细胞。使用 Gr-1 单克隆抗体清除 C57BL/6 小鼠中的髓源抑制细胞（MDSCs）。使用流式细胞术和免疫荧光检测肿瘤微环境中关键免疫细胞的变化。 结果：ETV4 的表达与人类 HCC 中的较高肿瘤-淋巴结-转移（TNM）分期、肿瘤分化不良、微血管侵犯和预后不良呈正相关。HCC 细胞中 ETV4 的过表达反式激活了 PD-L1 和 CCL2 的表达，增加了肿瘤相关巨噬细胞（TAM）和 MDSC 的浸润，并抑制了 CD8 T 细胞的积累。慢病毒介导的 CCL2 敲低或 CCR2 抑制剂 CCX872 治疗削弱了 ETV4 诱导的 TAM 和 MDSC 浸润和 HCC 转移。此外，FGF19/FGFR4 和 HGF/c-MET 通过 ERK1/2 通路共同上调 ETV4 的表达。此外，ETV4 上调 FGFR4 的表达，下调 FGFR4 减少了 ETV4 增强的 HCC 转移，形成了 FGF19-ETV4-FGFR4 的正反馈回路。最后，抗 PD-L1 联合 FGFR4 抑制剂 BLU-554 或 MAPK 抑制剂 trametinib 显著抑制了 FGF19-ETV4 信号诱导的 HCC 转移。 结论：ETV4 是一种预后标志物，抗 PD-L1 联合 FGFR4 抑制剂 BLU-554 或 MAPK 抑制剂 trametinib 可能是抑制 HCC 转移的有效策略。 影响和意义：在这里，我们报告 ETV4 增加了 HCC 细胞中 PD-L1 和趋化因子 CCL2 的表达，导致 TAM 和 MDSC 的积累和 CD8 T 细胞的抑制，从而促进 HCC 转移。更重要的是，我们发现抗 PD-L1 联合 FGFR4 抑制剂 BLU-554 或 MAPK 抑制剂 trametinib 显著抑制了 FGF19-ETV4 信号介导的 HCC 转移。这项临床前研究将为开发 HCC 患者新的联合免疫治疗策略提供理论依据。
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