Background: Genetic variants (MTTP, TM6SF2, FABP1) that modulate hepatic steatosis are linked to increased hepatocellular carcinoma (HCC). Liver specific deletion of Mttp (Mttp-LKO) or Tms6sf2 increase fibrosis, steatosis and HCC, while Dgat2-LKO mice show decreased steatosis but not fibrosis. Fabp1 deletion in Mttp LKO mice (Fabp1 Mttp DKO) abrogated both hepatic steatosis and fibrosis.
Approach: How do upstream genetic modifiers (Fabp1, Dgat2) alter tumorigenesis in mice with impaired VLDL secretion? Mice were injected with diethylnitrosamine (DEN) and fed chow or high fat fructose cholesterol (HFC) diet for up to 12 months.
Results: DEN-injected Mttp LKO mice exhibited increased tumor burden vs Mttp^f/f on either chow or HFC diet (Fig 1A), with increased steatosis and fibrosis (Fig 1B). Fabp1 Mttp DKO mice on chow exhibited increased tumor area at 6m (LKO, 2.9 ± 1.3 % tumor; DKO, 8.0 ± 2.2, n=11, p=0.07) and 12m (Fig 1C), with increased mortality (<40% survival) and HCC (Fig 1C). Histology showed increased steatotic tumors in Mttp LKO livers (vs Fabp1 Mttp DKO) with increased focal necrosis and increased inflammation in DKO tumors (Fig 1D). In contrast, mice fed HFC diet exhibited similar tumor burden (LKO, 1.5 ± 0.5 % tumor; DKO, 0.7 ± 0.5, n=9-11; ns) suggesting altered energy availability/utilization enhances tumor growth in Fabp1 Mttp DKO mice. Consistent with this, expression of glycolytic genes (Pkm2/Hk2) were increased in Fabp1 Mttp DKO livers, and DKO hepatocytes showed increased ability to use glucose and glutamine as fuel (Seahorse assay, Fig 1E). This suggests that metabolic adaptations triggered by Fabp1 deletion may shift tumor morphology and energy utilization, contributing to increased tumor growth. HFC fed Dgat2 LKO mice exhibit reduced hepatic steatosis and combined Dgat2/Mttp deletion (Dgat2 Mttp DKO) abrogated steatosis and decreased fibrogenic gene expression (Fig 2A). In DEN-injected, HFC-fed mice Dgat2 deletion reduced tumor burden in Mttp LKO mice at 4m (LKO, 1.5 ± 0.7 % tumor; DKO, 0.26 ± 0.12, n=10-11, p=0.14) and 7 months (Fig 2B), with decreased fibrotic area and hepatocyte proliferation (Fig 2C). RNA seq revealed reduced expression of genes related to cholesterol metabolism (Pcsk9, Lss, Cyp51) in Dgat2 Mttp DKO livers vs Flox control (Fig 2D), suggesting alterations in cholesterol and fatty acid metabolism influence tumor phenotype.
Conclusions: Altered expression of upstream pathways (Fabp1 Dgat2) in lipid droplet turnover and utilization modulate the impact of impaired VLDL secretion in experimental tumorigenesis. Fabp1 deletion mitigates fibrosis in HF fed mice but alters energy utilization to promote tumor growth in chow fed animals. Dgat2 deletion attenuates both steatosis and tumorigenesis, suggesting it may be a viable target for HCC prevention.

Figure 1: Germline Fabp1 deletion increases tumor burden in Mttp LKO mice with alterations in tumor phenotype and energy utilization. A. Hepatic tumor area, expressed as percent of total tissue area in Mttp LKO mice fed chow or HFC. B. Hepatic triglyceride (TG, left) and PSR-stained fibrotic area in DEN treated Mttp LKO mice (HFC diet, 4 months). C. Tumor area (left) and prevalence of HCC in H&E stained liver tissue in chow diet fed mice at 12 months. D. Histological analysis of tumor phenotype in chow fed mice at 12 months. Slides were scored by a trained pathologist blinded to genotype. n=7 Flox, 6 Fabp1-/-, 9 Mttp LKO, 9 Fabp1 Mttp DKO. Representative images (100x magnification) are shown. E. Seahorse assays evaluating dependence on and capacity of isolated primary hepatocytes to utilize distinct energy sources. For all panels, * p<0.05; ** p<0.01; **** p<0.005.

Figure 2: Deletion of Dgat2 reduces steatosis, fibrosis and tumor burden in DEN-injected Mttp LKO mice fed HFC diet. A. Hepatic TG (left) and fibrogenic gene expression in female mice fed HFC diet for 6 months (no DEN). B. Tumor area (left) and hepatic TG in DEN-treated, HFC fed mice at 7 months. C. Fibrotic area (left) and hepatocyte proliferation (% BrdU positive nuclei, expressed as % of hepatocyte nuclei) at 4 months. D. Hepatic expression of genes related to cholesterol metabolism at 4 months. n=5/genotype. For all panels, * p<0.05; ** p<0.01; *** p<0.005, **** p<0.001.

Xiuli Liu

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