Hormonally Induced Hepatocellular Carcinoma in Diabetic Wild Type and Carbohydrate Responsive Element Binding Protein Knockout Mice.

OBJECTIVE: In the rat, the pancreatic islet transplantation model is an established method to induce hepatocellular carcinomas (HCC), due to insulin-mediated metabolic and molecular alterations like increased glycolysis and de novo lipogenesis and the oncogenic AKT/mTOR pathway including upregulation of the transcription factor Carbohydrate-response element-binding protein (ChREBP). ChREBP could therefore represent an essential oncogenic co-factor during hormonally induced hepatocarcinogenesis. METHODS: Pancreatic islet transplantation was implemented in diabetic C57Bl/6J (wild type, WT) and ChREBP-knockout (KO) mice for 6 and 12 months. Liver tissue was examined using histology, immunohistochemistry, electron microscopy and Western blot analysis. Finally, we performed NGS-based transcriptome analysis between WT and KO liver tumor tissues. RESULTS: Three hepatocellular carcinomas were detectable after 6 and 12 months in diabetic transplanted WT mice, but only one in a KO mouse after 12 months. Pre-neoplastic clear cell foci (CCF) were also present in liver acini downstream of the islets in WT and KO mice. In KO tumors, glycolysis, de novo lipogenesis and AKT/mTOR signalling were strongly downregulated compared to WT lesions. Extrafocal liver tissue of diabetic, transplanted KO mice revealed less glycogen storage and proliferative activity than WT mice. From transcriptome analysis, we identified a set of transcripts pertaining to metabolic, oncogenic and immunogenic pathways that are differentially expressed between tumors of WT and KO mice. Of 315 metabolism-associated genes, we observed 199 genes that displayed upregulation in the tumor of WT mice, whereas 116 transcripts showed their downregulated expression in KO mice tumor. CONCLUSIONS: The pancreatic islet transplantation model is a suitable method to study hormonally induced hepatocarcinogenesis also in mice, allowing combination with gene knockout models. Our data indicate that deletion of ChREBP delays insulin-induced hepatocarcinogenesis, suggesting a combined oncogenic and lipogenic function of ChREBP along AKT/mTOR-mediated proliferation of hepatocytes and induction of hepatocellular carcinoma.

Hepatocellular glycogenotic foci after combined intraportal pancreatic islet transplantation and knockout of the carbohydrate responsive element binding protein in diabetic mice.

AIMS: The intraportal pancreatic islet transplantation (IPIT) model of diabetic rats is an insulin mediated model of hepatocarcinogenesis characterized by the induction of clear cell foci (CCF) of altered hepatocytes, which are pre-neoplastic lesions excessively storing glycogen (glycogenosis) and exhibiting activation of the AKT/mTOR protooncogenic pathway. In this study, we transferred the IPIT model to the mouse and combined it with the knockout of the transcription factor carbohydrate responsive element binding protein (chREBP). METHODS: C57BL/6J Wild-type (WT) and chREBP-knockout (chREBP-KO) mice (n = 297) were matched to 16 groups (WT/ chREBP-KO, experimental/control, streptozotocine-induced diabetic/not diabetic, one/four weeks). Experimental groups received the intraportal transplantation of 70 pancreatic islets. Liver and pancreatic tissue was examined using histology, morphometry, enzyme- and immunohistochemistry and electron microscopy. RESULTS: CCF emerged in the liver acini downstream of the transplanted islets. In comparison to WT lesions, CCF of chREBP-KO mice displayed more glycogen accumulation, reduced activity of the gluconeogenic enzyme glucose-6-phosphatase, decreased glycolysis, lipogenesis and reduced levels of the AKT/mTOR cascade members. Proliferative activity of CCF was ∼two folds higher in WT mice than in chREBP-KO mice. CONCLUSIONS: The IPIT model is applicable to mice, as murine CCF resemble preneoplastic liver lesions from this hepatocarcinogenesis model in the rat in terms of morphological, metabolic and molecular alterations and proliferative activity, which is diminished after chREBP knockout. chREBP appears to be an essential component of AKT/mTOR mediated cell proliferation and the metabolic switch from a glycogenotic to lipogenic phenotype in precursor lesions of hepatocarcinogenesis.

Inter-laboratory comparison of turkey in ovo carcinogenicity assessment (IOCA) of hepatocarcinogens.

In three independent laboratories carcinogens (diethylnitrosamine, DEN, 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone, NNK) and non-carcinogens (N-nitrosoproline, nicotine) were evaluated in turkey eggs for in ovo carcinogenicity assessment (IOCA). Compounds were injected into aseptic fertilized eggs. After incubation for 24 days, foci of altered hepatocytes (FAH), some with a pseudoglandular structure and/or signs of compression of the surrounding tissue were observed in the fetal liver. All laboratories were able to distinguish unequivocally the hepatocarcinogen-exposed groups from those exposed to non-carcinogens or the vehicle controls, based on the pre-specified evaluation parameters: tumor-like lesions, pseudoglandular areas and FAH. In addition to focal changes, only the carcinogens induced hepatocellular karyomegaly. Lower doses of the carcinogens, which did not induce FAH, were sufficient to induce hepatocellular karyomegaly. After exposure to 4 mg DEN, gall bladder agenesis was observed in all fetuses. The IOCA may be a valuable tool for early investigative studies on carcinogenicity and since it does not use rodents may complement chronic rat or mouse bioassays. Test substances that are positive in both rodents and fertilized turkey eggs are most probably trans-species carcinogens with particular significance for humans. The good concordance observed among the three laboratories demonstrates that the IOCA is a reliable and robust method.