Objective   To explore the mechanism underlying E26 transformation-specific homologous factor (EHF) in regulating the stemness of pancreatic cancer cells.

  Methods   Bioinformatic analyses were conducted to identify the downstream pathway regulating the stemness of pancreatic cancer cells through EHF. Western blot and RT-qPCR were used to verify the correlation between EHF expression and the activity of the selected stemness pathway in pancreatic cancer. ChIP and double luciferase reporter gene experiments were used to verify the function of EHF in regulating the target genes. After treatment with pathway inhibitors, functional experiments were performed to determine the importance of the selected pathway in the EHF-mediated regulation of pancreatic cancer stemness.

  Results   Bioinformatic analyses revealed that the Wnt/β-catenin pathway mediates the stemness of pancreatic cancer cells through EHF regulation. Pancreatic cells with stably overexpressed or downregulated EHF were constructed. Further Western bolt and RT-qPCR verified that EHF expression was negatively correlated to the activity of the Wnt/β-catenin pathway in pancreatic cancer cells (P<0.01). The direct binding of EHF to the promoter region of β-catenin could ultimately inhibitits transcription. After the treatment with XAV939, an inhibitor of the Wnt/β-catenin pathway, functional experiments including flow cytometry, suspension cell spheroidization, and soft agar clone formation indicated that the upregulation of stemness characteristics caused by EHF knockdown could be effectively inhibited (P<0.01) in pancreatic cancer.

  Conclusions   EHF inhibits the stemness of pancreatic cancer cells by blocking Wnt/β-catenin pathway. Small molecule inhibitors targeting Wnt/β-catenin pathway have the potential to treat pancreatic cancer in future.