Introduction: Obesity and type 2 diabetes are risk factors for liver cancer due to a transition from steatosis to nonalcoholic steatohepatitis (NASH), resulting in liver fibrosis. If left untreated, liver fibrosis can progress into cirrhosis leading to hepatocellular carcinoma (HCC). Connective tissue growth factor (CTGF) is a pro-fibrotic matricellular protein that is highly expressed during hepatocarcinogenesis. This study aims to investigate the involvement of CTGF in nonalcoholic steatohepatitis (NASH) and liver cancer development during diabetic conditions induced by streptozotocin (STZ) treatment and the feeding of a high fat diet (HFD) in mice.
Methods: Transgenic male mice expressing green fluorescent protein (GFP) under the control of Ctgf promoter (Ctgfp-GFP), liver specific CTGF knockouts (CtgfΔhep/Δhep), and control mice that contained two alleles of floxed Ctgf (Ctgffloxed/floxed) were given STZ (200 ng/pup) at postnatal day 2 (P2) through subcutaneous injection and were fed HFD at postnatal week four to induce NASH and HCC. The treated animals were harvested at postnatal week 5, 12, and 20 for early steatosis, fibrosis and liver cancer development respectively. CTGF expression was analyzed by immunofluorescent staining, Western analysis, and qRT-PCR. In addition, the mouse liver cancer RT2 profiler PCR array was screened to identify cancer-related genes that were differentially expressed during NASH and HCC development comparing CtgfΔhep/Δhep and Ctgffloxed/floxed mice.
Results: All three types of animals developed NASH and HCC in response to STZ and HFD feeding. Liver pathologies ranging from steatosis, NASH, liver fibrosis, and nodule formation, to intratumoral angiogenesis were observed as discrete molecular and histological stages in the STZ/HFD induced NASH-HCC model. Immunofluorescent analysis of Ctgfp-GFP reporter mice showed induction of the Ctgf gene in vascular endothelial cells, biliary epithelial cells, hepatocytes, and inflammatory cells in STZ/HFD livers. We utilized the mouse liver cancer RT2 profiler PCR array and compared the expression of 90 liver cancer related genes between CtgfΔhep/Δhep and Ctgff/f tumors that developed after 12-week HFD feeding. 12 upregulated genes and 10 downregulated genes were identified in Ctgfk/k mice compared to Ctgff/f animals.
Conclusion: We successfully established a NASH-HCC model combining STZ and HFD treatment. CTGF expression was found in multiple cell types including endothelial cells, cholangiocytes, inflammatory cells, and hepatocytes. Liver specific deletion of CTGF was associated with differential expression of groups of genes involved in cell proliferation and apoptosis. The functional relationship between these genes and CTGF in liver cancer development in the setting of metabolic syndrome needs to be characterized in future studies.
Citation Format: Liya Pi, Marda Jorgensen, Seh-Hoon Oh, Altin Gjymishka, Bryon E. Petersen. The involvement of connective tissue growth factor in nonalcoholic steatohepatitis and liver cancer development under diabetic condition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5107.