ZG16 represses tumor progression and M2 polarization of tumor-associated macrophages in hepatocellular carcinoma by promoting ubiquitination and degradation of SNX9 via binding to ITCH.

backgroundHepatocellular carcinoma (HCC) is a highly aggressive primary liver malignancy characterized by limited therapeutic options and poor prognosis. Within the tumor microenvironment (TME), tumor-associated macrophages (TAMs) predominantly exhibit an M2-like phenotype, contributing to immune escape and tumor progression. Zymogen granule protein 16 (ZG16) has been reported to be downregulated in HCC, but its precise biological function and molecular mechanisms remain poorly understood. Therefore, we aimed to investigate the impact of ZG16 on HCC cell metastasis and TAM infiltration, as well as to elucidate its molecular mechanism.

methodsGain- and loss-of-function assays were used to verify the effect of ZG16 on HCC cell metastasis, as well as the recruitment and M2 polarization of TAMs. The underlying mechanism of ZG16 was explored by immunoprecipitation-liquid chromatography-mass spectrometry (IP-LC/MS) analysis, co-immunoprecipitation (co-IP) assay, and GST pull-down assay.

resultsOur results demonstrated that ZG16 overexpression significantly inhibited metastasis of HCC cells while also suppressing the recruitment and M2 polarization of TAMs, suggesting its dual role in both tumor cell-intrinsic and microenvironmental regulation. Notably, sorting nexin 9 (SNX9), a facilitator of HCC, was identified as a downstream target of ZG16. Mechanistically, we uncovered that ZG16 physically interacted with SNX9 and promoted its protein degradation through the ubiquitin-proteasome pathway. Functional rescue experiments provided compelling evidence that SNX9 overexpression effectively counteracted ZG16-mediated suppression of both HCC progression and TAM M2 polarization. Further mechanism exploration confirmed that ZG16 promoted the ubiquitination and degradation of SNX9 by recruiting itchy E3 ubiquitin protein ligase (ITCH).

conclusionsOur findings certify that ZG16 suppresses tumor progression and M2 polarization of TAMs in HCC through ITCH-mediated ubiquitination and subsequent degradation of SNX9. The ZG16/ITCH/SNX9 axis may represent an important regulatory pathway and potential therapeutic target for HCC.