Oncogenic mutations and dysregulated pathways in obesity-associated hepatocellular carcinoma

Abstract

Epidemiological studies showed that obesity and its related non-alcoholic fatty liver disease (NAFLD) promote hepatocellular carcinoma (HCC) development. We aimed to uncover the genetic alterations of NAFLD-HCC using whole-exome sequencing. We compared HCC development in genetically obese mice and dietary obese mice with wild-type lean mice fed a normal chow after treatment with diethylnitrosamine. HCC tumor and adjacent normal samples from obese and lean mice were then subjected to whole-exome sequencing. Functional and mechanistic importance of the identified mutations in Carboxyl ester lipase (Cel) gene and Harvey rat sarcoma virus oncogene 1 (Hras) was further elucidated. We demonstrated significantly higher incidences of HCC in both genetic and dietary obese mice with NAFLD development as compared with lean mice without NAFLD. The mutational signatures of NAFLD-HCC and lean HCC were distinct, with <3% overlapped. Eight metabolic or oncogenic pathways were found to be significantly enriched by mutated genes in NAFLD-HCC, but only two of these pathways were dysregulated by mutations in lean HCC. In particular, Cel was mutated significantly more frequently in NAFLD-HCC than in lean HCC. The multiple-site mutations in Cel are loss-of-function mutations, with effects similar to Cel knock-down. Mutant Cel caused accumulation of cholesteryl ester in liver cells, which led to induction of endoplasmic reticulum stress and consequently activated the IRE1α/c-Jun N-terminal kinase (JNK)/c-Jun/activating protein-1 (AP-1) signaling cascade to promote liver cell growth. In addition, single-site mutations in Hras at codon 61 were found in NAFLD-HCC but none in lean HCC. The gain-of-function mutations in Hras (Q61R and Q61K) significantly promoted liver cell growth through activating the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDK1)/Akt pathways. In conclusion, we have identified mutation signature and pathways in NAFLD-associated HCC. Mutations in Cel and Hras have important roles in NAFLD-associated hepatocellular carcinogenesis.

Figure 1

Obesity increases susceptibility of hepatocarcinogenesis in mice. (a) Schematic illustration of the treatment of wild-type (WT) control lean mice (n=10), genetically obese (db/db) mice (n=13) and dietary obese HFD mice (n=10). Mice were killed at 7 months of age. Body weight, liver weight and liver-to-body weight ratio were examined. (b) Representative gross morphology of liver tumors and microscopic features of HCC in H&E-stained liver sections were shown. (c) HCC incidence, number of HCCs per mouse and maximal size of the tumors were examined. All comparisons are performed by unpaired t tests except incidence (Fisher's exact test). *P<0.05, **P<0.001. T, tumor; N, non-tumor. Means±s.e.m. are shown in the bar charts, while means±s.d. are indicated in the corresponding ‘Result'.

Figure 2

Whole-exome sequencing analysis identified genomic alterations of NAFLD-HCC. (a) HCC-associated non-synonymous mutations were identified by exome sequencing in db/db mice and WT lean mice. Genes mutated in more than one case were defined as recurrently mutated genes. (b) KEGG pathway enrichment analysis identified eight important pathways of interest to be significantly dysregulated in db/db obese mice, while only two in lean mice.

Figure 3

Mutations in Cel and growth-promoting effect of loss of CEL. (a) Schematic illustration of the somatic non-synonymous mutations in Cel identified in liver tumors from obese mice (16 genetic, 13 dietary) and control lean mice (n=16). (b) The mRNA expression of CEL in nutrition-associated HCC (n=78) was significantly lower than in virus infection-associated HCC (n=56) that was available in TCGA database (independent t-test). (c) Stable knock-down of CEL in normal hepatocyte cell line MIHA by two shCEL vectors (SH1 and SH2) was confirmed by real-time PCR and western blot. (d) CEL downregulation in MIHA cells increased cell growth as shown by MTT assay. (e) CEL downregulation in MIHA cells promoted colony formation ability of MIHA cells. Data are mean±s.e.m. in (b) and mean±s.d. elsewhere. *P<0.05, **P<0.01. NC, negative control shRNA; SH1&SH2, shRNAs targeting CEL.

Figure 4

Loss of CEL caused intracellular accumulation of cholesteryl ester to induce ER stress and activation of IRE1α/JNK/c-Jun/AP-1 pathway. (a1) CEL knock-down increased the level of cholesteryl ester in MIHA cells. (a2) Treatment with 25 nm cholesteryl palmitate, a form of cholesteryl ester having liquid-crystalline states, promoted cell growth as shown by MTT assay. (b1) CEL knock-down in MIHA cells activated the signaling cascade in ER stress as shown by western blot analysis of key proteins. (b2) CEL knock-down increased the transcriptional activity of AP-1 in MIHA as determined by luciferase reporter assay. (c1) MIHA cells expressing CEL mutants (Mut-1, Mut-2) caused significantly higher levels of cholesteryl ester as compared with cells overexpressing wild-type CEL. (c2) CEL mutants promoted MIHA cell growth as shown by MTT assay. (d1) CEL mutants activated the signaling cascade of ER stress in MIHA. (d2) CEL mutants increased the transcriptional activity of AP-1 in MIHA. (e) Cholesteryl ester levels (e1) and protein levels of p-IRE1a and p-c-Jun (e2) in NAFLD-HCC and lean HCC from mouse models. Proteins from two lean HCC were not adequate for both tests. (f) Mechanistic scheme of CEL knock-down or inactivating mutation in promoting liver cancer growth. Data are means±s.d. *P<0.05; **P<0.01; ***P<0.001. NC, negative control shRNA; SH1&SH2, shRNAs targeting CEL.

Figure 5

Hras mutants promoted cell proliferation. (a) Somatic non-synonymous mutations in Hras (all located at codon 61) were found in NAFLD-HCCs of one dietary and two genetically obese mice but in none of the control lean mice. (b) Hras mutants (Q61R and Q61K) promoted cell proliferation compared with wild-type (WT) Hras and control vector (Ctrl) transfection in MIHA cells by colony formation assay. (c) Hras mutants (Q61R and Q61K) promoted cell viability compared with WT Hras and control vector transfection in MIHA cells by MTT assay. (d) Mutations of Q61R or Q61K enhanced the Ras activity of Hras. (e) Hras mutants (Q61R and Q61K) increased the protein expression of key regulators of Ras/MAPK and PI3K/AKT signaling cascades. (f) Protein level of p-p85 was examined by western blot in NAFLD-HCC and lean HCC from mouse models. The same bands for GAPDH were shown as in Figure 4e2 for the same protein samples. (g) Mechanistic scheme of signaling cascade mediated by wild-type and mutant Hras. Data were expressed as mean±s.d. *P<0.05, **P<0.01 and ***P<0.001 as compared with control vector transfection; ^#P<0.05 and ^##P<0.01 as compared with wild-type Hras.

Figure 6

Schematic summary of this study. Genes harboring non-synonymous somatic mutations were identified in NAFLD-HCC mice, affecting eight cancer signaling pathways. Among all mutated genes, Cel and Hras are of particular interest. Mutations in these two genes both have oncogenic effects with different mechanisms.