Dietary cholesterol promotes steatohepatitis related hepatocellular carcinoma through dysregulated metabolism and calcium signaling.

The underlining mechanisms of dietary cholesterol and nonalcoholic steatohepatitis (NASH) in contributing to hepatocellular carcinoma (HCC) remain undefined. Here we demonstrated that high-fat-non-cholesterol-fed mice developed simple steatosis, whilst high-fat-high-cholesterol-fed mice developed NASH. Moreover, dietary cholesterol induced larger and more numerous NASH-HCCs than non-cholesterol-induced steatosis-HCCs in diethylnitrosamine-treated mice. NASH-HCCs displayed significantly more aberrant gene expression-enriched signaling pathways and more non-synonymous somatic mutations than steatosis-HCCs (335 ± 84/sample vs 43 ± 13/sample). Integrated genetic and expressional alterations in NASH-HCCs affected distinct genes pertinent to five pathways: calcium, insulin, cell adhesion, axon guidance and metabolism. Some of the novel aberrant gene expression, mutations and core oncogenic pathways identified in cholesterol-associated NASH-HCCs in mice were confirmed in human NASH-HCCs, which included metabolism-related genes (ALDH18A1, CAD, CHKA, POLD4, PSPH and SQLE) and recurrently mutated genes (RYR1, MTOR, SDK1, CACNA1H and RYR2). These findings add insights into the link of cholesterol to NASH and NASH-HCC and provide potential therapeutic targets.

Deriving and testing of dysplastic murine hepatocytes: A new platform in liver cancer research.

Dysplastic hepatocytes (DH) represent altered hepatocytes with potential for malignant transformation. To date, most research on pathways to hepatocarcinogenesis has focused on use of "hepatoma" cell lines derived from hepatocellular carcinoma (HCC). We describe a novel technique for deriving/culturing DH and demonstrate their utility for functional studies in vitro, compared to primary hepatocytes (PH) and HCC. PH and DH were prepared by portal vein collagenase perfusion from C57BL/6J mice. DH were subsequently subjected to FACS. HCC from diethylnitrosamine (DEN)-injected mice were mechanically isolated. Cell cycle analyses were performed by flow cytometry and PCNA immunohistochemistry. To establish utility of DH, we studied pathways of p53 turnover, apoptosis and cell proliferation using pfithrin-α (PFT) and nutlin-3. Like PH, DH were minimally proliferative compared to HCC. Only 30±0.03% of DH were in G2/M phase versus 51±0.01% of HCC; this difference corroborated with PCNA-immunostaining of dysplastic nodules from DEN-injected mice. In DH and HCC, nutlin-3 suppressed p53 mRNA, induced p53 and mdm2 activation but paradoxically resulted in increased anti-apoptotic and proliferative activity. Primary murine DH display distinctive biological characteristics compared with PH and HCC. As an intermediate cell type to HCC, they offer a new pathobiologically relevant primary cell culture system with which to interrogate the molecular changes in hepatocarcinogenesis.

Obesity and diabetes accelerate hepatocarcinogenesis via hepatocyte proliferation independent of NF-κB or Akt/mTORC1.

BACKGROUND: There are strong links between obesity, diabetes and hepatocellular carcinoma (HCC), but molecular mechanisms remain unclear. AIM: We tested the proposed involvement of NF-κB, IL-6/STAT3 and Akt/mTORC1 before onset (at 3 months) and at onset (6 months) of accelerated hepatocarcinogenesis in DEN-injected obese and diabetic foz/foz compared to lean wildtype (Wt) mice, and also studied the hepatocyte proliferative response to DNA damage between the obese and lean lines. METHODS: Male foz/foz and Wt littermates fed normal chow were DEN-injected (10mg/kg i.p.) at age 12-15 days. To test the effect of mTOR inhibitor on growth of dysplastic hepatocytes, a separate cohort of DEN-injected foz/foz mice was administered rapamycin (4 mg/kg body weight/day). RESULTS: foz/foz mice developed obesity, hyperinsulinemia, diabetes, adipokine dysregulation and fatty liver, without increased serum or liver TNF-α or serum IL-6. All DEN-injected foz/foz mice developed HCC by 6 mths vs. 0/10 lean Wt. At 3 mths, there were more dysplastic hepatocytes in DEN-injected foz/foz than Wt, with increased liver injury (serum ALT), hepatocyte apoptosis (M30-positive cells) and proliferation (cyclin D1, cyclin E, PCNA), but neither NF-κB nor STAT3 activation. foz/foz livers exhibited upregulation of DNA damage sensors ATM and ATR, with inadequate cell cycle checkpoint controls (CHK1, CHK2, p53, p21). Akt and mTORC1 were highly activated in livers from foz/foz vs. Wt mice. Despite such activation, rapamycin failed to reduce growth of dysplastic hepatocytes. CONCLUSIONS: Accelerated DEN-induced HCC in obese/diabetic mice is linked to enhanced growth of dysplastic hepatocytes that cannot be attributed to NF-κB or IL-6/STAT3 activation, nor to sustained mTORC1 activation. The critical mechanism for obesity-enhanced hepatocarcinogenesis lies in the disconnection between hepatocellular injury with DNA damage, and an unrestrained proliferative response. RELEVANCE FOR PATIENTS: This study supports the epidemiological data linking obesity, diabetes and fatty liver disease with increased risk for developing HCC. The findings also suggest that mTORC1 inhibition may not be beneficial in the prevention of obesity-related hepatocarcinogenesis.

Neutrophil-derived myeloperoxidase aggravates non-alcoholic steatohepatitis in low-density lipoprotein receptor-deficient mice.

BACKGROUND: Chronic inflammation and oxidative stress play fundamental roles in the pathogenesis of non-alcoholic steatohepatitis (NASH). Previously, we reported that myeloperoxidase (MPO), an aggressive oxidant-generating neutrophil enzyme, is associated with NASH severity in man. We now investigated the hypothesis that MPO contributes to the development and progression of NASH. METHODOLOGY: Low-density lipoprotein receptor-deficient mice with an MPO-deficient hematopoietic system (LDLR(-/-/)MPO(-/-tp) mice) were generated and compared with LDLR(-/-/)MPO(+/+tp) mice after induction of NASH by high-fat feeding. RESULTS: High-fat feeding caused a ~4-fold induction of liver MPO in LDLR(-/-/)MPO(+/+) mice which was associated with hepatic sequestration of MPO-positive neutrophils and high levels of nitrotyrosine, a marker of MPO activity. Importantly, LDLR(-/-/)MPO(-/-tp) mice displayed markedly reduced hepatic neutrophil and T-lymphocyte infiltration (p<0.05), and strong down regulation of pro-inflammatory genes such as TNF-α and IL-6 (p<0.05, p<0.01) in comparison with LDLR(-/-/)MPO(+/+tp) mice. Next to the generalized reduction of inflammation, liver cholesterol accumulation was significantly diminished in LDLR(-/-/)MPO(-/-tp) mice (p = 0.01). Moreover, MPO deficiency appeared to attenuate the development of hepatic fibrosis as evident from reduced hydroxyproline levels (p<0.01). Interestingly, visceral adipose tissue inflammation was markedly reduced in LDLR(-/-/)MPO(-/-tp) mice, with a complete lack of macrophage crown-like structures. In conclusion, MPO deficiency attenuates the development of NASH and diminishes adipose tissue inflammation in response to a high fat diet, supporting an important role for neutrophils in the pathogenesis of metabolic disease.