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

Abstract

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.

Fig. 1

Cholesterol augmented high-fat (HF) diet in accelerating hepatocarcinogenesis in mice. a Schematic illustration of the treatment of mice. Male C57BL/6J mice were administered a single injection of the carcinogen diethylnitrosamine (DEN) intraperitoneally at age 15 days and fed normal chow (n = 9), HF (n = 10) or high-fat high-cholesterol (HFHC; n = 10) diets starting from 6 weeks of age till 8 months. b Body weights of mice fed HF or HFHC diets were similar and both significantly higher than normal chow (NC)-fed mice. c Fasting blood glucose and intraperitoneal glucose tolerance test (IPGTT) levels in mice fed NC, HF or HFHC diets. *P < 0.05. d Mice fed HFHC diet had significantly higher liver weights and liver-body weight ratios than HF-fed mice. e Levels of hepatic triglyceride, hepatic free cholesterol and cholesterol ester, and serum cholesterol were measured in HF- and HFHC-fed mice. f, g Representative H&E staining histological images (f) and NAFLD scores (g) of liver tissues from HF- and HFHC-fed mice. h HCC incidence, number and size in NC-, HF- and HFHC-fed mice after DEN injection. Tumor number: few < 5; multiple ≥ 5. Tumor size: small, all < 5 mm³; large, at least one ≥ 5 mm³. The data are shown as means ± SE for IPGTT and means ± SD for others. Data in b-g between each two groups were compared using ANOVA Tukey’s multiple comparison tests

Fig. 2

Expressional aberration of inflammation-related genes associated with NASH development in HFHC-fed mice. a Clustering of differentially expressed genes in HFHC-induced NASH as compared to HF-induced steatosis. b Pathways enriched by differentially expressed genes in NASH vs. steatosis. #gene, the number of annotated genes in the input list / number of annotated genes in the reference list. adjP, p value adjusted by multiple test adjustment. c Differentially expressed inflammatory genes in NASH compared to steatosis. Expression levels were normalized to the mean level of each gene among all samples shown. d Schematic illustration of the dysregulated pathways involved in the development of HFHC-induced NASH

Fig. 3

Aberrant gene expression and the dysregulated pathways in HCCs from HF- and HFHC-fed mice. a Heat map of differentially expressed genes in HCCs versus adjacent non-tumorous samples in HF- and HFHC-fed mice. With the majority in both groups upregulated, the two groups shared about half of the upregulated genes. b Pathways dysregulated commonly in both HFHC-associated NASH-HCCs and HF-associated steatosis-HCCs, or specifically in one group, by aberrant gene expression (≥4 genes involved, P < 0.05 by multiple test adjustment) are shown. Pathways highlighted in red were also dysregulated in NASH versus steatosis. c Differentially expressed genes in HCCs compared to adjacent non-tumorous livers (upper panel, unique in HFHC-fed mice; lower panel, common in both groups). Expression levels were normalized to the mean level of each gene among all samples shown

Fig. 4

Aberrant gene expression verified in human NASH-HCC samples as compared to adjacent non-tumor livers. a Shown are RNA levels in FPKM in 17 pairs of tumors compared to adjacent non-tumor livers from patients with NASH-HCC. P values by paired t tests and FDR adjusted by Benjamini–Hochberg method are shown. Expression levels were normalized to the mean level of each gene among all samples shown. b Expression of seven genes was further validated to be significantly altered in 12 pairs of NASH-HCCs (T) compared to adjacent non-tumor livers (N) by RT-qPCR. Comparison by paired t tests

Fig. 5

NASH-HCCs from HFHC-fed mice harbored significantly more mutations than steatosis-HCCs from HF-fed mice. a Circos indication of somatic mutations identified by whole-exome-sequencing. b Numbers of somatic mutations in each sample classified according to mutation types. *only 1 indel was found in one NASH-HCC and thus was counted in non-synonymous mutations. Comparison of the total and non-synonymous mutation numbers showed significant difference between HF and HFHC groups (unpaired t tests). c Distribution of genes recurrently mutated (in ≥2 samples) in each group and commonly mutated in both groups (non-synonymous). Numbers and percentages of genes recurrently mutated are shown in each group. HF, high-fat diet; HFHC, high-fat high-cholesterol diet

Fig. 6

Pathways dysregulated by genetic alterations. a Seven pathways were significantly dysregulated in three or more NASH-HCCs from HFHC-fed mice ( ≥ 3 genes involved in each sample, adjusted P < 0.05). b Mutated genes in calcium signaling pathway are indicated. 28 genes were mutated in NASH-HCCs, 6 of which were recurrently mutated. Only one gene in calcium signaling pathway was mutated in one of the steatosis-HCCs. Color-filled icons show mutated genes. Recurrently mutated genes were highlighted with affected NASH-HCC numbers denoted. c Mutated genes in insulin signaling are indicated. 16 gene were mutated in NASH-HCCs, 2 of which were recurrently mutated, while no gene was mutated in steatosis-HCCs. d Five pathways were enriched commonly by mutations and aberrant gene expression in NASH-HCCs

Fig. 7

Validation of recurrently mutated genes in human NASH-HCCs. a Recurrently mutated genes identified in mouse NAHS-HCCs were checked in 37 human NASH-HCCs (17 in-house and 20 from TCGA). Recurrent mutations in 21 genes were verified in human NASH-HCCs. b Recurrently mutated calcium signaling-related genes in human NASH-HCCs. c Mutation frequencies of eight genes were higher in HCCs with risk history of NAFLD than others without NAFLD (Chi-square test). *P < 0.05 and **P < 0.001 for all NAFLD-HCCs (in-house and TCGA, n = 37) versus other HCCs; ^#P < 0.05 for NAFLD-HCCs from TCGA (n = 20) vs. other HCCs; ns non-significant as compared with other HCCs. d Schematic illustration of the mutation sites in Ryr1 identified in mouse and human NASH-HCCs

Fig. 8

Schematic summary of this study. NASH development promoted by dietary cholesterol is associated with aberrant gene expression linked with activated inflammatory signaling, dysregulated metabolic and cancer-related pathways. Dietary cholesterol-induced NASH-HCC development in mice is associated with aberrant gene expression, genomic mutations and the associated core pathways. The identified aberrant gene expression and mutations were verified in human NASH-HCCs. NC, normal chow; HF, high-fat diet; HFHC, high-fat high-cholesterol diet