Abstract
Background: Hepatocellular carcinoma (HCC) is an aggressive liver cancer with poor prognosis. Deubiquitinating enzymes (DUBs) are critical regulators of tumor progression, yet the functional significance of DUBs in HCC remains poorly understood. Methods: HCC patient-derived organoids (PDOs), HCC cell lines and animal models were used to evaluate the anticancer responses of ubiquitin-specific protease (USP)2 inhibition. We analyzed the correlation of USP2 expression and immune cells infiltration using single-cell RNA sequencing and flow cytometry analysis. Mechanistically, we established an in vitro co-culture system and analyzed metabolic data to find out the bridge between tumor cell USP2 and macrophage in the microenvironment. Immunofluorescence, co-immunoprecipitation, CUT&RUN, ELISA, and mass spectrometry were conducted to explore the molecular pathway. Results: We found that the inhibitor (ML364) targeting USP2 shows effective anticancer responses against HCC PDOs. Targeting USP2 significantly inhibits lipid metabolism of HCC and induces cell ferroptosis. Single-cell RNA sequencing analysis and multiplex immunohistochemistry analysis indicated that high expression of USP2 in HCC was associated with the infiltration of M2 macrophage. Mechanistically, USP2 deubiquitinates and stabilizes peroxisome proliferator-activated receptor gamma (PPARγ) via removing the K48-linked ubiquitin chain at the K142 site. PPARγ promotes the transcription of fatty acid biosynthesis-related genes (ATP-citrate lyase, acetyl-CoA carboxylase and ACSS2) and de novo synthesis of fatty acids including oleic acid. HCC cell-derived oleic acid promotes M2 macrophage polarization by enhancing the fatty acid oxidation of macrophages. Polarized M2 macrophages further secrete interleukin-10, which created an IL-10/STAT3/USP2 positive-feedback loop to activate USP2 expression continuously. Conclusion: Our data suggest that USP2, a key molecule mediating the interaction between HCC cells and tumor-associated macrophages, may be a promising therapeutic target for HCC.