Tumor-derived ITIH2 activates PI3K\AKT pathway via THBS1 ubiquitination and promotes tumor angiogenesis in hepatocellular carcinoma.

The heterogeneous tumor microenvironment (TME) plays a critical role in the initiation and progression of hepatocellular carcinoma (HCC). Within the TME, tumor vascular endothelial cells (ECs) are key stromal components that drive angiogenesis. However, the molecular mechanisms of interaction relationships between angiogenesis and tumor progression in HCC remain unclear. We performed clustering analysis on scRNA-seq data from two HCC patients (GSE166635) and identified eight distinct cell types using uniform manifold approximation and projection (UMAP). An eight-gene risk signature was established to predict patient prognosis. Using iterative least absolute shrinkage and selection operator (LASSO) and multivariate Cox regression, we developed a risk scoring system and constructed a clinically applicable nomogram with TCGA HCC dataset as training cohort and ICGC HCC dataset as validation cohort. The predictive performance of the model was assessed and validated through calibration and decision curve analysis (DCA). Furthermore, we identified ITIH2 as a novel prognostic biomarker in HCC that may be associated with angiogenesis. Immunohistochemical (IHC) staining of clinical samples verified the prognostic value of ITIH2, which was also correlated with the levels of angiogenesis biomarkers, CD31 and CD34. Interestingly, functional experiments, including colony formation, CCK-8, and flow cytometry assays, revealed that ITIH2 overexpression did not alter tumor cell proliferation or apoptosis. Instead, ITIH2 enhanced the angiogenic capacity of ECs and promoted tumor progression in both in vitro and in vivo experiments. However, overexpression of THBS1 could reverse the function of ITIH2 on ECs and PI3K/AKT signaling pathway. Mechanistically, tumor-derived ITIH2 was associated with activation of the PI3K/AKT signaling pathway in ECs via a ubiquitination-dependent regulation of THBS1. The findings recapitulated that tumor-derived ITIH2 could promote HCC progression by enhancing the angiogenic ability of ECs by activating PI3K/AKT signaling pathway via THBS1 stability mediated by ubiquitination.