Dipeptidyl peptidases in experimental hepatocellular carcinoma

Abstract

The urgent unmet need to develop hepatocellular carcinoma (HCC) therapies is addressed here by characterising a novel mouse model of HCC in the context of ongoing liver damage and overnutrition. Male C57Bl/6J mice were treated with Diethylnitrosamine (DEN) and thioacetamide (TAA), and some were provided with an atherogenic high fat diet (HFD). Inflammation, steatosis, fibrosis, 87 genes, liver lesions and intratumoural leukocyte subsets were quantified up to 24 weeks of age. Mice treated on the DEN/TAA/HFD regime for 24-36 weeks were then treated with one of several compounds, ARI-4175, ARI-3996, ARI-6000 or vehicle controls. These compounds target the DPP4 enzyme family. The DPP4 enzyme family includes four atypical serine proteases: DPP4, Fibroblast activation protein (FAP), DPP8, and DPP9. These proteins mediate a diverse range of biological processes by releasing the N-terminal dipeptide of substrates that have a proline or alanine at the penultimate position. Livers were assessed for tumour and histopathological burdens. The expression and activity of DPP4, FAP, DPP8 and DPP9 were investigated to assess their biomarker utility and roles in liver carcinogenesis. Adding HFD to DEN/TAA increased fibrosis, steatosis and inflammation, and the incidence of both HCC and non-HCC dysplastic lesions. All lesions contained α-SMA positive fibroblasts. Macrophage marker F4/80 was not significantly different between treatment groups, but the macrophage-associated genes Arg-1 and Cd47 were differentially expressed. Fibrosis, cancer and cell death associated genes were upregulated in DEN/TAA/HFD livers. Fewer Kupffer cells and plasmacytoid dendritic cells were in tumours compared to control liver. FAP+ fibroblastic populations can be identified with a novel reagent 4613b FAP+ fibroblastic populations were acutely depleted from livers with FAP specific pro-drug ARI-3996. Depletion of FAP+ fibroblast populations with FAP-specific prodrugs ARI-3996 and ARI-6000 as a result of long term administration did not decrease liver fibrosis. ARI-6000 treatment showed a nonsignificant trend towards less inflammation and fibrosis. ARI-4175 administration increased liver fibrosis and decreased tumour burden in the liver. Liver DPP4, DPP8, and DPP9 enzymatic activity expression decreased with DEN/TAA/HFD treatment and DPP9 was enriched in tumours and dysplastic lesions. In conclusion, combining hepatotoxins with an atherogenic diet produced more intrahepatic tumours, dysplastic lesions and fibrosis compared to hepatotoxins alone. This new HCC model provides a relatively rapid means of examining primary HCC and potential therapies in the Preface xii context of multiple hepatotoxins including those derived from overnutrition. The DPP-family have shown potential as both HCC markers and potential therapeutic targets due to their tissue specific expression in the case of FAP and roles in liver carcinogenesis in the case of DPP4 and DPP9.