Deubiquitinase OTUD5 facilitates stiffness-induced hepatic stellate cell activation by stabilizing YAP

Hepatic stellate cells (HSCs) activate and transdifferentiate into tumor-promoting myofibroblasts, which is an essential cause of colorectal liver metastasis. In addition to cytokines such as transforming growth factor-β, matrix stiffness is also a vital factor for promoting HSC activation. Our recent study reveals that OTU deubiquitinase 5 (OTUD5) promotes hepatocellular carcinoma progression, but its role and molecular mechanism in stiffness-induced HSC activation remain unclear. Polyacrylamide hydrogels with different composition ratios were used to simulate different matrix stiffnesses. Western blotting and immunofluorescence were performed to detect OTUD5, α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), Yes-associated protein (YAP), cysteine-rich angiogenic inducer 61 (CYR61), HA, FLAG, ubiquitin (UB), and MYC levels. RT-qPCR was used to assess the expression of OTUD5, YAP, CYR61, and CTGF mRNA. The interaction between OTUD5 and YAP, as well as the YAP ubiquitination level, was determined using a co-immunoprecipitation assay. The proliferation and invasion of HT-29 cells were determined by CCK-8 and transwell assays. The nude mouse subcutaneous co-injection model was used to evaluate the growth of HT-29 cells in vivo. HSCs cultured on a stiff substrate (11 kPa) showed significantly higher OTUD5 expression compared to those in the soft substrate (2 kPa). OTUD5 knockdown prominently attenuated stiffness-induced HSC activation. The stiffness-induced increase in OTUD5 expression was significantly abolished by YAP knockdown in HSCs. YAP overexpression enhanced, while YAP knockdown and inhibition reduced OTUD5 expression. In turn, OTUD5 positively regulated YAP protein level and YAP target genes, such as CYR61 and CTGF, in HSCs. Co-immunoprecipitation and immunofluorescence assays confirmed the interaction between OTUD5 and YAP. Mechanistically, OTUD5 functioned as a deubiquitinase, removing the K48 ubiquitin chain from YAP and maintaining protein stability. Functionally, OTUD5 knockdown markedly attenuated HSCs-induced HT-29 cell proliferation and invasion in vitro and tumor growth in vivo. Our results highlight the critical role of OTUD5/YAP feedback loop in stiffness-induced HSC activation, offering a potential therapeutic target for colorectal liver metastasis.