Super-enhancer-driven AJUBA is activated by TCF4 and involved in epithelial-mesenchymal transition in the progression of Hepatocellular Carcinoma

Background and Aims: Aberrant transcriptional programs are highly regulated processes that play important roles in the development and progression of hepatocellular carcinoma (HCC). Emerging evidence suggests that super-enhancers (SEs) often drive critical oncogene expression. However, SE-associated genes in HCC pathogenesis are still poorly understood. Methods: We performed integrative ChIP-seq and Hi-C analyses of HCC cells and identified ajuba LIM protein (AJUBA) as a SE-associated gene. We evaluated AJUBA expression in HCC using immunohistochemistry, immunoblotting, and qRT-PCR. ChIP and luciferase reporter assays were performed to demonstrate that transcription factor 4 (TCF4) bound to AJUBA-associated SEs. We then assessed the role of AJUBA in HCC using both in vitro and in vivo assays. Epithelial-mesenchymal transition (EMT) was examined using immunofluorescence and immunoblotting assays. Furthermore, we used immunoprecipitation and BiFC assays to explore the underlying mechanisms. Results: We identified AJUBA as a SE-associated oncogene in HCC regulated by TCF4. High AJUBA expression was related to an aggressive phenotype and unfavorable outcome in HCC patients. AJUBA knockdown significantly reduced cell migration and invasion capacities both in vitro and in vivo. Furthermore, AJUBA overexpression in HCC recruited tumor necrosis factor associated factor 6 (TRAF6), enhancing the phosphorylation of Akt and increasing Akt activity toward GSK-3β, thus promoting EMT. Conclusions: Our results provide functional and mechanistic links between the SE-associated gene AJUBA and tumor EMT in aggressive HCC.