Project description:Abstract Rationale: Interferon (IFN) plays a key role in immunotherapy by acting through interferon-stimulated genes. Interferon resistance limits its efficacy. Ubiquitination modification of key proteins is a popular research direction in hepatocellular carcinoma (HCC), and it is also one of the important mechanisms of interferon resistance. This study is dedicated to discovering the link between the pathogenesis of interferon resistance and ubiquitination modifications in HCC. Methods: We first combined the TCGA LIHC dataset and genes from the iUUCD 2.0 database to screen the most promising ubiquitination-related gene, UBE2O, in HCC using a bioinformatics approach. A combination of proteomic analysis, mass spectrometry, and survival analysis was used to screen for pathways and proteins negatively regulated by UBE2O. The effect of silencing UBE2O on interferon sensitivity in HCC cells was assessed using in vitro clone formation, migration, and wound healing assays and in vivo subcutaneous tumor xenograft models. Changes in the half-life of substrates following inhibition of UBE2O were assessed using a cycloheximide method. Two databases were used to predict ubiquitination sites on IFIT3. After rescue experiments to exclude the effect of substrate, proteomic analysis was reperformed to analyze the effect on the interferon pathway and phenotype. The effect on substrate and interferon sensitivity was observed using arsenic trioxide (ATO) to inhibit UBE2O. Results: Initially, UBE2O was deduced from 807 UUCRGs in the TCGA LIHC dataset by six consecutive screening steps. UBE2O was significantly overexpressed in HCC cell lines and tissues compared to the corresponding normal group, and low expression of UBE2O was associated with a better prognosis. UBE2O was knocked down for proteomic analysis, and the results suggested that UBE2O negatively regulates the interferon-alpha/beta signaling (p＜0.01) and the interferon pathway (p＜0.01). IFIT3 in the interferon pathway was identified as a possible ubiquitinated substrate of UBE2O by combining mass spectrometry, the human protein atlas (HPA), and TCGA-based survival analysis. There was a significant negative correlation between UBE2O and IFIT3, including survival analysis showing opposite trends and a negative correlation in protein expression. UBE2O binds to IFIT3, and interferon-α promotes this binding. The stability of IFIT3 protein increased with decreasing UBE2O (p＜0.05), and these effects disappeared with the mutational inactivation of UBE2O. K236 of IFIT3 is the active site of ubiquitination. By increasing IFIT3, the knockdown of UBE2O increases the sensitivity of HCC cells to interferon, leading to reduced proliferation and migration (all p＜0.05). These effects disappeared after the elimination of IFIT3. Additionally, ATO inhibited UBE2O and therefore increased interferon sensitivity in HCC cells. Conclusions: UBE2O exerts a protumor role and targets a new substrate, IFIT3, for ubiquitination and degradation, impeding the interferon pathway activation and interferon sensitization in HCC. The findings will shed light on the interferon-based targeted or combination therapeutic strategies for HCC in the future.

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SF3A3 regulates the proliferation and migration capabilities of hepatocellular carcinoma cells via the Notch signaling pathway

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