The Role of Fructose in Hepatocarcinogenesis

Abstract

Worldwide and in Australia hepatocellular carcinoma (HCC) is one of the fastest growing malignancies in terms of incidence, and in Australia has a dismal 5 year survival rate of 16%. Thus, there is an urgent and unmet need for better therapeutics to treat this devastating disease. The growing epidemic of obesity parallels hyper-nutrition that is associated with a Western diet, which in turn promotes non alcoholic fatty liver disease (NAFLD) and the more severe phenotype non alcoholic steatohepatitis (NASH), a major cause of the increasing incidence of HCC. A major component of the western diet is the consumption of sugars, especially the natural sweetener fructose that due to its cheapness and stability, is used in preference to glucose. In comparison to glucose, fructose is metabolised differently, and studies have linked fructose consumption to hepatic inflammation, NAFLD/NASH and increased HCC risk and growth in rodents. Furthermore, being overweight and obese is linked with low serum levels the fat-derived protein adiponectin, that are associated with insulin resistance, increased prevalence of NAFLD and NASH, liver fibrosis, and from our previous studies, increased liver tumour growth. Therefore, adiponectin is one of the crucial mediators of hepatic malignancies. Given that fructose consumption is increasing, we therefore hypothesised that the combination of increased fructose digestion and low serum adiponectin would promote aggressive HCC growth. To test this, we used two experimental models: (i) the A52 NASH HCC transplantation model developed in our laboratory, and (ii) the mutagen diethylnitrosamine (DEN) HCC model using wild-type (WT) and adiponectin knock-out mice (APN KO); fed either a normal chow (NC) or high fructose (HF) diet. Unexpectedly, we observed that a HF delayed A52 tumour formation, and in vivo the serine to glycine synthesis (SGS) and pentose phosphate pathways (PPP) were upregulated. Moreover, in wild-type HF fed hosts subcutaneous A52 tumours evolved differently, they had increased the expression of the methyltransferase G9a, oncoprotein c-Myc and the gluconeogenic enzyme fructose 1,6 bisphosphatase (FBP1), and reduced keratin-19 a marker of less differentiated tumour cells. Given the upregulated metabolic pathways, we chose to restrict growth in vitro, through the application of the inhibitors BIX-01294 and MB05032, that target G9a, and indirectly the SGS, and FBP1, respectively. The application of each inhibitor alone reduced A52 tumour cell proliferation, and their combined application ablated mitosis at least 2-fold more. These data suggest that the in vivo application of these inhibitors could be a plausible way to treat NASH HCC. To further test the role of fructose in a genetic model, we treated 15 day old WT and APN KO mice with DEN, and from 6 weeks of age fed them a NC or HF diet. At 20 weeks it was found in both genotypes that the HF fed mice had a lower basal glucose, and after a pyruvate challenge test the HF APN KO mice had reduced glucose levels, suggesting an impaired gluconeogenic response. 9 months after DEN injection the mice were euthanised, and in agreement with our previous data NC fed APN KO mice had liver tumours 2.7-fold larger than NC-fed WT mice. Surprisingly, there was no difference in tumour size between NC and HF groups in WT mice. Remarkably, HF APN KO liver tumours were 29.7-fold smaller than tumours in NC fed APN KO mice. Histology confirmed reductions in cellular proliferation in the tumour and non-tumour regions of HF APN KO mice. There were limited changes in fibrotic, inflammatory and lipogenic markers between groups. Western blot analyses illustrated that the HF APN KO livers have down regulated mTOR signaling in comparison to NC APN KO mice, suggesting a mechanism through growth is impaired. To characterise the genetic changes associated with APN loss and a NC or HF diet on tumour growth, Next Generation Sequencing was performed. This identified >190 genes significantly dysregulated and four novel gene targets were identified: Ribosomal protein L41 (RPL41), Carbonyl reductase 3 (Cbr3), HORMA domain containing 2 (Hormad2) and Solute Carrier Family 5 (Sodium/Myo-Inositol Cotransporter) Member 3 (SLC5A3). In conclusion, in this work we find unexpected attributes of a HF diet on HCC growth, namely that subcutaneous tumour growth is delayed and is associated with altered metabolism and tumour type, and in genetic model reduced through the inhibition of growth signals and the induction of a novel genetic program. The characterization of these processes will lead to the identification of novel targets for treatment of this poorly treated malignancy.