Transcription factor JUNB is required for transformation of EpCAM-positive hepatocellular carcinoma (HCC) cells into CD90-positive HCC cells in vitro

Hepatocellular carcinoma (HCC) harbors two types of stem cells—epithelial and mesenchymal stem cells. The mechanism by which epithelial EpCAM-positive HCC cells transform into mesenchymal CD90-positive HCC cells remains unclear. On peritumoral fibrotic nodules, epithelial HCC cells form communities with stromal cells, driving tumor growth and malignancy. We aimed to clarify the mechanism by which epithelial cell adhesion molecule (EpCAM)-positive HCC cells contribute to the phenotype of mesenchymal CD90-positive HCC cells that metastasize to distant sites by elucidating the interaction between EpCAM-positive HCC cells and fibroblasts. We investigated the changes in cell surface markers and gene expression in cocultures of EpCAM-positive CD90-negative epithelial HCC cells (Huh1, Huh7, patient HCC cells) and fibroblasts (Lx-2 and Tig3-20) using fluorescence-activated cell sorting and quantitative real-time polymerase chain reaction. Furthermore, we performed whole transcriptome RNA-seq analysis of cocultured epithelial HCC cells to identify the transcription factors responsible for the phenotypic changes, and verified the acquisition of metastatic ability by subcutaneous injection of these cells in mice. By coculturing epithelial HCC cells and fibroblasts, we evaluated the rate of transformation to mesenchymal CD90-positive HCC cells in vitro and in vivo, and examined the expression of epithelial–mesenchymal transition-related genes. We found that epithelial EpCAM-positive hepatoma cells were transformed into mesenchymal CD90-positive hepatoma cells, exhibiting metastatic potential, by the transcription factor JUNB. The JUNB expression in ​​EpCAM-positive hepatoma cells was increased by paracrine stimulation with fibroblast-derived TGFb1. This study unravels the mechanism by which fibroblasts aggravate the malignancy of liver cancer, and the results suggest that JUNB may be a target for treating liver cancer metastasis.