Cell-permeated peptide P-T3H2 inhibits malignancy on hepatocarcinoma through stabilizing HNF4α protein

Objectives: Hepatocyte nuclear factor 4α (HNF4α) is a key regulator of hepatocyte function and has a strong therapeutic effect on hepatocellular carcinoma (HCC) by inducing the differentiation of hepatoma cell into hepatocytes. Our previous study showed that Tribbles homolog 3 (TRIB3) directly interacts with and promotes the degradation of HNF4α in non-alcoholic fatty liver disease (NAFLD). Disrupting the TRIB3-HNF4α interaction by using a cell-permeating peptide called P-T3H2 stabilizes HNF4α protein. This study aimed to assess the anti-tumor impact of P-T3H2 in HCC. Methods: The expression of TRIB3 and HNF4α was assessed using western blot and immunohistochemistry staining (IHC). Hepatic functions in HCC cells were evaluated through periodic acid-Schiff (PAS) staining and acetylated low-density lipoprotein (ac-LDL) uptake. Senescence-associated β-galactosidase (SA-β-gal) activity staining was used to detected the cellular senescence in HCC cells. RNA-Seq analysis was carried out to identify differentially expressed genes in Huh-7 cells treated with the peptide P-T3H2 compared to the control peptide P-T3H2-2A. The impact of P-T3H2 on hepatocarcinoma malignancy was evaluated in HCC cells, a Huh-7 xenograft mouse model, an orthotopic model, and a C-MET/ΔN90-β-catenin-driven HCC model. Results: TRIB3 exhibited a negative correlation with HNF4α in both human and mouse HCC tissues. The administration of P-T3H2 significantly inhibited the malignancy of HCC cells. Additionally, P-T3H2 stabilized the HNF4α protein and facilitated the restoration of hepatic functions and the cellular senescence in HCC cells. RNA-Seq analysis demonstrated that P-T3H2 enhanced the transcriptional activity of HNF4α in HCC. Furthermore, P-T3H2 effectively suppressed the growth of HCC tumors in both of the Huh7 xenograft and orthotopic mouse models in vivo. Notably, P-T3H2 also inhibits hepatocarcinogenesis in the C-MET/ΔN90-β-catenin-driven HCC model.