Background: Stiffened extracellular matrix is a primary risk factor for hepatocellular carcinoma (HCC). We aim to investigate the mechanism by which matrix stiffness promotes malignant proliferation and develop an experimental therapeutic strategy for HCC. Methods: CCl 4 -induced and ATP-binding cassette subfamily B member 4 ( Abcb4 ) -/- spontaneous liver-fibrosis mouse models were used to validate the tumour-promoting effect of matrix stiffness in HCC. Polyacrylamide gel was used to simulate matrix with varying stiffness. RNA sequencing (RNA-seq), in vitro proliferation assays, and in vivo tumour models were used to elucidate the role of mechanically responsive protein ankyrin repeat domain protein 1 (ANKRD1) in HCC. Biochemical assays were performed to reveal the interaction between ANKRD1 and protein arginine methyltransferase 5 (PRMT5). Chromatin immunoprecipitation and luciferase reporter assays were performed to confirm the pattern of transcriptional regulation. The anti-tumour efficiency of Hippo pathway inhibitors and the PRMT5 inhibitor was evaluated in vitro and in vivo . Results: Analysis of human hepatic fibrosis index, fibrotic mice bearing HCC, and polyacrylamide gel-based soft matrix culture model demonstrated that a stiff microenvironment promoted HCC progression. RNA-seq of HCC cells cultured on varying stiffness conditions identified ANKRD1 as a target for mechanical signaling, which was further validated in human HCC tissue samples at both RNA and protein levels. Gene function analysis revealed that ANKRD1 promoted HCC progression, which partially mediated stiffness-associated malignant proliferation. Mechanistically, protein-protein interaction assays and quantitative post-translational modification assays proved the interaction between the C-terminus of ANKRD1 and PRMT5, which promoted the cytoplasmic localization of PRMT5 and arginine methylome remodeling. Furthermore, inhibitors of the Hippo pathway decreased ANKRD1 expression and sensitized HCC cells to PRMT5 inhibitor LLY-283 in both an in vitro cytotoxicity assay and a murine fibrotic HCC model. Conclusions: ANKRD1 is a mechanically responsive protein that promotes the proliferation of HCC cells by regulating PRMT5 activity. The combination of Yes1-associated transcriptional regulator/tafazzin (YAP/TAZ) inhibitors and a PRMT5 inhibitor is a potential therapeutic strategy for HCC patients with a stiffened tumour microenvironment.
