Polyubiquitination and accumulation of PEG10 regulated by SIAH1/2 affiliates the progression of hepatocellular carcinoma

BACKGROUND: SIAH1 and SIAH2 are key regulators of hepatocellular carcinoma (HCC) initiation and progression, but their precise molecular mechanisms remain unclear. Although both interact with the oncoprotein PEG10, it is unknown whether PEG10 mediates their regulatory effects on HCC or what the underlying mechanisms are. METHODS: The effects of SIAH1/2 on HCC cell proliferation and metastasis were assessed in vitro and in vivo by EdU, colony formation, transwell assays, and tumor xenograft models. Co-immunoprecipitation, ubiquitination assays, and site-directed mutagenesis were applied to investigate the interaction with PEG10 and the specific ubiquitination mechanisms. Clinical samples were used for analyzing the correlation between SIAH1/2 and PEG10 protein expression in HCC patients. RESULTS: We demonstrated that SIAH1 and SIAH2 played opposing roles in HCC: SIAH1 suppressed cell proliferation and metastasis, whereas SIAH2 promoted these processes. Mechanistically, both proteins bound to the PAIR domain of PEG10 and promoted its polyubiquitination via K48/63-linked chains. However, they differed in lysine specificity: SIAH1 mainly mediated K48-linked ubiquitination at Lys 36 and K63-linked ubiquitination at Lys 170 of PEG10, whereas SIAH2 primarily mediated K48-linked ubiquitination at Lys 36, and K63-linked ubiquitination at Lys 19 and Lys 155. In vivo analyses confirmed that SIAH1 expression negatively correlated with PEG10 protein levels, while SIAH2 showed a positive correlation. CONCLUSIONS: Our study reveals that SIAH1 and SIAH2 differentially regulate HCC progression by modulating PEG10 stability through distinct ubiquitination site targeting. These findings elucidate a key molecular mechanism in HCC and highlight the SIAH-PEG10 axis as a potential therapeutic target.