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. 2017 Oct;6(10):2370-2384.
doi: 10.1002/cam4.1177. Epub 2017 Sep 21.

Metabolomic and transcriptomic profiling of hepatocellular carcinomas in Hras12V transgenic mice

Tingting Fan  1 , Zhuona Rong  1 , Jianyi Dong  1 , Juan Li  1 , Kangwei Wang  1 , Xinxin Wang  1 , Huiling Li  1 , Jun Chen  1 , Fujin Wang  1 , Jingyu Wang  1 , Aiguo Wang  1
Affiliations

Metabolomic and transcriptomic profiling of hepatocellular carcinomas in Hras12V transgenic mice

Tingting Fan et al. Cancer Med. 2017 Oct.

Abstract

Activation of the Ras/MAPK pathway is prevalently involved in the occurrence and development of hepatocellular carcinoma (HCC). However, its effects on the deregulated cellular metabolic processes involved in HCC in vivo remain unknown. In this study, a mouse model of HCC induced by hepatocyte-specific expression of the Hras12V oncogene was investigated using an integrative analysis of metabolomics and transcriptomics data. Consistent with the phenotype of abundant lipid droplets in HCC, the lipid biosynthesis in HCC was significantly enhanced by (1) a sufficient supply of acetyl-CoA from enhanced glycolysis and citrate shuttle activity; (2) a sufficient supply of NADPH from enhanced pentose phosphate pathway (PPP) activity; (3) upregulation of key enzymes associated with lipid biosynthesis; and (4) downregulation of key enzymes associated with bile acid biosynthesis. In addition, glutathione (GSH) was significantly elevated, which may result from a sufficient supply of 5-oxoproline and L-glutamate as well as an enhanced reduction in the process of GSSG being turned into GSH by NADPH. The high level of GSH along with elevated Bcl2 and Ucp2 expression may contribute to a normal level of reactive oxygen species (ROS) in HCC. In conclusion, our results suggest that the lipid metabolism, glycolysis, PPP, tricarboxylic acid (TCA) cycle, citrate shuttle activity, bile acid synthesis, and redox homeostasis in the HCC induced by ras oncogene are significantly perturbed, and these altered metabolic processes may play crucial roles in the carcinogenesis, development, and pathological characteristics of HCC.

Keywords: Hepatocellular carcinoma; Ras oncogene; metabolomics; transcriptomics.

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Figures

Figure 1
Figure 1
Gross anatomic and histopathological analysis of hepatic alterations. (A) Representative liver stereogram image of a liver from a 9‐month‐old wild‐type male. (B) Representative liver stereogram image of a liver carrying multiple tumors from a 9‐month‐old Ras‐Tg male. The red arrows indicate the hepatic alterations. (C) Relative histopathological H&E staining images (100 × ) showing liver tissue from a wild‐type mouse. (D) Relative histopathological H&E staining images (100 × ; upright corner: 400 × ) showing HCC from a Ras‐Tg mouse. W: wild‐type liver tissue; T: hepatocellular carcinoma (HCC).
Figure 2
Figure 2
Score plots of PCA, PLS‐DA, and OPLS‐DA based on the metabolite profile data of HCC and wild‐type liver tissues. (A) Score plots of PCA based on the metabolite profile data for wild‐type liver tissues (black circles) and HCC (blue rhombi). The principal components PC1 (t[1]) and PC2 (t[2]) described 33.6% and 15.6% of the variation, respectively (n = 8). (B) The plot of PLS‐DA scores showing almost complete separation of wild‐type liver tissues (black circles) and HCC (blue rhombi). The classification parameters were R2X (cum) = 0.539, R2Y (cum) = 0.951, and Q2 (cum) = 0.746. (C) Validation model of PLS‐DA for wild‐type liver tissues (blue squares) and HCC (green squares) for 200 permutations of the data showing the degradation of R2 to below 0.515 and Q2 to below −0.173. (D) OPLS‐DA showing the contribution of variables to the difference between wild‐type liver tissues (black circles) and HCC (blue rhombi). The classification parameters were R2X (cum) = 0.816, R2Y (cum) = 0.996, and Q2 (cum) = 0.874.
Figure 3
Figure 3
Pathway enrichment analysis for significantly changed genes and metabolites. (A) The significantly upregulated genes and (B) downregulated genes in HCC compared to wild‐type liver tissues were analyzed by KEGG pathway enrichment assays. (C) Significantly changed metabolites in HCC compared to wild‐type liver tissues were analyzed by MetaboAnalyst version 3.0 for metabolite set enrichment. The p values for the metabolic pathways are color coded, with dark black representing the most significant values and white representing the least significant values. Detailed information is shown in Tables [Link], [Link], [Link], [Link]. M.: metabolism.
Figure 4
Figure 4
Schematic representations of the most relevant metabolic and transcriptional differences between HCC and wild‐type liver tissues. Metabolic pathways related to glycolysis, the TCA cycle, PPP, and lipid biosynthesis. (B) Metabolic pathways related to glutathione. (C) Metabolic pathways related to cholesterol and bile acid synthesis. Red indicates significantly higher concentrations of metabolites, expression levels of enzymes, or enhanced pathways in HCC; green indicates significantly lower concentrations of metabolites, expression levels of enzymes, or attenuated pathways in HCC; and gray indicates unchanged or undetermined results. The standard letters indicate metabolites, the italic letters indicate enzymes, and the underlined italic letters indicate pathways. The metabolites with broken circles indicate the central metabolites in HCC. (D) The reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), pyruvate, lactate, cholesterol (TC), triglyceride (TG), ROS, GSH, and bile acid (BA) was detected in wild‐type liver (W) and HCC (T) tissues using the methods described in the Materials and Methods section. The data are expressed as the mean ± SEM (n = 8). *P < 0.05, **P < 0.01, and ***P < 0.001.
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