FGF19/FGFR4-mediated elevation of ETV4 facilitates hepatocellular carcinoma metastasis by upregulating PD-L1 and CCL2

Abstract

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.

Keywords: Anti-PD-L1; BLU-554; CCX872; Myeloid-derived suppressor cell; Tumour-associated macrophage.