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Squalene epoxidase drives NAFLD-induced hepatocellular carcinoma and is a pharmaceutical target

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Science Translational Medicine  18 Apr 2018:
Vol. 10, Issue 437, eaap9840
DOI: 10.1126/scitranslmed.aap9840
  • Fig. 1 SQLE is overexpressed in NAFLD-HCC.

    (A) RNA-seq analysis of 18 paired NAFLD-HCC and adjacent normal tissues (left). SQLE was the top outlier gene among the up-regulated metabolic genes (right). FPKM, fragments per kilobase of transcript per million mapped reads. (B) SQLE mRNA expression in 17 individual paired NAFLD-HCC and adjacent normal samples (one paired sample was not available for analysis). (C) Increased SQLE mRNA and (D) protein expression in human NAFLD-HCC were validated in an independent cohort. Arrows show SQLE protein. (E) Sqle mRNA expression was up-regulated in dietary and genetic NAFLD-HCC animal models: DEN-injected and high-fat diet–treated wild-type (WT) mice (left) and DEN-treated db/db mice (right). (F) Analysis of correlation between SQLE gene copy number and mRNA expression in 17 paired NAFLD-HCC. Data are means ± SEM. (C to E) Paired two-tailed Student’s t tests were used. (F) The Pearson correlation coefficient was used.

  • Fig. 2 SQLE promotes NAFLD-HCC cell growth.

    (A) Overexpression of SQLE in LO2 (normal liver) and HKCI10 (NAFLD-HCC) cells was confirmed by Western blot analysis. Ectopic SQLE expression promoted cell viability (A) and colony formation (B) (n = 3, performed in triplicate). (C) SQLE knockdown in HKCI2 (NAFLD-HCC) cells was confirmed by Western blot analysis. SQLE knockdown suppressed cell viability (C) and colony formation (D) (n = 3, performed in triplicate). (E) Cells were stained with propidium iodide (PI) and analyzed by flow cytometry. Flow cytometry analysis indicated that SQLE expression decreased the number of cells in G1 phase but increased the number of cells in S phase (n = 3, performed in triplicate). (F) Cells were stained with PI and analyzed by flow cytometry. Flow cytometry analysis indicated that knockdown of SQLE in HKCI2 induced G1 arrest (n = 3, performed in triplicate). (G) Western blot analysis indicated that SQLE overexpression increased PCNA and cyclin D1 expression, whereas SQLE knockdown had the opposite effects. (H) SQLE overexpression reduced apoptosis, as determined by annexin V–phycoerythrin and 7-aminoactinomycin D (7-AAD) staining and flow cytometry (n = 3, performed in triplicate). (I) SQLE knockdown in HKCI2 cells induced apoptosis, as determined by annexin V–phycoerythrin and 7-AAD staining and flow cytometry (n = 3, performed in triplicate). (J) Western blot analysis showed that SQLE overexpression reduced the protein expression of cleaved forms of caspase-7 and caspase-9, whereas SQLE knockdown had the opposite effects. Data are means ± SEM. Difference in cell viability between two groups was determined by repeated-measures ANOVA. Mann-Whitney U test or Student’s t test was performed to compare the variables in two groups (colony formation, cell cycle, and apoptosis). *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 3 Hepatocyte-specific transgenic SQLE expression in mice accelerates HFHC diet–associated NAFLD-HCC.

    (A) Scheme for the generation of mice with hepatocyte-specific Sqle overexpression. Western blot confirmed overexpression of SQLE in the livers of Sqle tg mice. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) Experimental design for DEN-injected and HFHC diet–induced mouse models of NAFLD-HCC. At the age of 10 to 13 days, WT or Sqle tg mice were injected with a single dose of DEN. Starting at 6 weeks of age, mice were fed with HFHC diet until week 25 (top). H&E staining of WT and Sqle tg mouse livers (middle). HCC tumor incidence and multiplicities in WT and Sqle tg mice (bottom). The red arrows show the tumor. Results are means ± SEM (n = 9 to 10). (C) Hepatocyte-specific Sqle expression increased liver weight (left) and liver/body weight ratio (middle), but not body weight (right), in DEN-injected HFHC diet–treated mice. (D) Serum AFP (left), ALT (middle), and AST (right) concentrations of WT and Sqle tg mice treated with DEN and HFHC diet. Results are means ± SEM (n = 9 to 10). (E) Ki-67 staining of livers from DEN-injected and HFHC diet–treated WT and Sqle tg mice. The red arrows show the Ki-67–positive cells. T, tumor; N, adjacent normal. Results are means ± SEM (n = 9 to 10). Mann-Whitney U test was used. Scale bars, 50 μm. ***P < 0.001.

  • Fig. 4 SQLE promotes intracellular cholesterol/cholesteryl ester accumulation, which induces tumor cell growth.

    (A) Hepatocyte-specific Sqle expression increased liver free cholesterol (left) and cholesteryl ester (right) concentrations in normal diet or DEN-injected, HFHC diet–treated mice. (B) SQLE overexpression in LO2 and HKCI10 cells increased intracellular free cholesterol and cholesteryl ester (left). Knockdown of SQLE in HKCI2 and an HCC cell line (BEL-7404) decreased intracellular free cholesterol and cholesteryl ester (right) (n = 4, performed in triplicate). (C) Exogenous cholesterol promoted cell growth and increased intracellular cholesteryl ester concentration but had no effect on free cholesterol (n = 3, performed in triplicate). (D) Avasimibe abolished the proliferative effect of cholesterol (n = 3, performed in triplicate). (E) Cholesteryl ester directly promoted cell growth (n = 2, performed in triplicate). (F) Avasimibe abolished SQLE-induced cell growth and (G) reversed cholesteryl ester accumulation (n = 4, performed in triplicate). Data are means ± SEM. The significance of the differences in cell growth rates was determined by repeated-measures ANOVA. The significance of the difference in cholesterol concentrations was determined by Mann-Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 5 Oncogenic function of SQLE depends on PTEN/PI3K/AKT/mTOR.

    (A) SQLE activated the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, as indicated by FOXO3 luciferase reporter assay (n = 3, performed in triplicate). FOXO3 is a target of AKT, and PI3K/AKT pathway activation inhibited FOXO3 expression. (B) PI3K/AKT pathway PCR analysis of LO2 cells overexpressing SQLE. PTEN is one of the outlier down-regulated genes. (C and D) Representative Western blot analysis confirmed that SQLE induced PTEN silencing and activated downstream oncogenic signaling [phosphorylated AKT (p-AKT) and phosphorylated mTOR (p-mTOR)] in vitro (C) and in vivo (D). (E) mRNA expression of PTEN/PI3K/AKT/mTOR downstream effectors in SQLE-overexpressing LO2 cells and SQLE-knockdown BEL-7404 cells (n = 3, performed in triplicate). (F) SQLE downstream effects on cholesteryl ester accumulation and cell growth depend on PTEN/mTOR. Knockdown of PTEN followed by overexpression of SQLE in LO2 and HKCI10 cells prevented the effects of SQLE on cholesteryl ester accumulation (left), cell viability (middle), and PTEN/mTOR signaling (right). (G) mTOR inhibitor rapamycin suppressed the effect of SQLE on cholesteryl ester accumulation (left) (n = 4, performed in triplicate) and cell growth (right) (n = 3, performed in triplicate). Data are means ± SEM. Mann-Whitney U test was used to assess the significance of the differences in cholesterol concentrations, mRNA expression, and luciferase reporter assay. The significance of the difference between cell growth rates was determined by repeated-measures ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 6 SQLE silences PTEN through ROS-mediated DNA hypermethylation.

    (A) SQLE increased NADP+/NADPH ratio in Sqle tg mice. (B) SQLE increased NADP+/NADPH ratio and ROS in LO2 and HKCI10 cells (n = 3, performed in triplicate). (C) GSH, a ROS scavenger, reversed SQLE-induced PTEN loss, as indicated by Western blot analysis. (D) GSH abolished SQLE-induced accumulation of cholesteryl ester in LO2 and HKCI10 cells (n = 4, performed in triplicate). (E to I) SQLE silenced PTEN via ROS-mediated DNMT3A expression. (E) Heat map of the Epigenetic Chromatin Modification Enzymes PCR Array using LO2 and HKCI2 cell lines overexpressing SQLE. (F) Quantitative PCR and Western blot confirmed that mRNA (top left) and protein (top right) expression of DNMT3A were positively regulated by SQLE. Nuclear DNMT3A activity was also induced by SQLE (bottom) (n = 3, performed in triplicate). (G) GSH reversed SQLE-induced DNMT3A expression. (H) Infinium HumanMethylation450 (450K) BeadChip analysis of CpG methylation in LO2-vector and LO2-SQLE cell lines revealed increased global promoter methylation in SQLE-overexpressing cells (left). Pathway analysis of hyper- and hypomethylated genes (right). (I) SQLE-induced PTEN silencing was reversed by DNMT3A knockdown (n = 3, performed in triplicate). (J) Schematic diagram showing the mechanism of action of SQLE in NAFLD-HCC. SQLE increases cholesterol biosynthesis and NADP+/NADPH-related ROS. Increased ROS induces DNMT3A expression and activation, which mediates transcriptional silencing of PTEN via promoter methylation. PTEN loss activates AKT/mTOR to promote NAFLD-HCC. AKT/mTOR activation also induces cholesteryl ester accumulation, which contributes to tumor cell growth. Data are means ± SEM. Mann-Whitney U test was used for comparing means between two groups. *P < 0.05, **P < 0.01.

  • Fig. 7 SQLE expression is increased in human HCC and correlates with poor survival.

    (A) SQLE mRNA expression in HCC was positively correlated with DNMT3A mRNA expression. The correlation analysis between DNMT3A and SQLE mRNA expression was performed for the NAFLD-HCC cohort (n = 16) and validated in our Guangzhou (n = 69), The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) (n = 423), and Stanford (n = 72) cohorts. Pearson correlation coefficient was used. (B and C) Representative Western blot confirmed that SQLE overexpression up-regulated DNMT3A expression and mTOR phosphorylation and also silenced PTEN expression in (B) NAFLD-HCC and HCC and (C) Sqle-induced mouse HCC. (D) SQLE gene expression in HCC and adjacent normal tissue was determined in our Guangzhou HCC cohort (n = 91 pairs) and (E) validated in TCGA-LIHC (n = 50 pairs) and Stanford cohorts (n = 65 pairs). Paired two-tailed Student’s t tests were used. Data are means ± SEM. (F and G) High SQLE expression correlates with poor survival in HCC. Kaplan-Meier survival analysis and Cox regression analysis of our cohort (high, n = 43; low, n = 45) (G), and TCGA-LIHC (high, n = 155; low, n = 175) cohort based on predictive survival analysis.

  • Fig. 8 SQLE inhibitor terbinafine suppresses NAFLD-HCC growth in vitro and in vivo.

    (A) Terbinafine treatment suppressed cell growth (left) and colony formation (right) in NAFLD-HCC (HKCI2, HKCI10) and HepG2 cell lines (n = 3, performed in triplicate). (B) Terbinafine suppressed SQLE expression and reversed the silencing of PTEN, as determined by Western blot (n = 3, performed in triplicate). (C) Terbinafine reduced the amounts of free cholesterol and cholesteryl ester in HCC cell lines (n = 4, performed in triplicate). (D) Terbinafine (80 mg/kg per day, oral) inhibited growth of subcutaneous HepG2 xenografts, as evidenced by reductions in tumor volume and weight. (E) Terbinafine increased the survival of mice harboring HepG2 xenografts. Kaplan-Meier analysis and log-rank test were used. (F) Terbinafine (80 mg/kg per day, oral) attenuated the growth of orthotopic HKCI2 xenografts. Both tumor volume and weight were reduced. (G) Terbinafine decreased the amounts of free cholesterol and cholesteryl ester in HepG2 xenografts and HKCI2 orthotopic nude mouse model. (H) Terbinafine (80 mg/kg per day, oral) suppressed tumorigenesis in DEN-injected and HFHC diet–treated Sqle tg mice (left), in terms of both tumor incidence and tumor number (right). (I) H&E and Ki-67 staining of vehicle and terbinafine-treated livers. The red arrows show the positive cells. Scale bars, 100 μm (H&E) and 50 μm (Ki-67). (J) Terbinafine treatment decreased liver/body weight ratio (left), liver and serum cholesterol concentrations (middle), and NADP+/NADPH ratio (right). (K) Representative Western blot analysis showed that terbinafine suppressed Sqle expression and reversed the effect of SQLE on downstream factors DNMT3A and PTEN. Data are means ± SEM. The significance of the difference between cell growth rates and tumor growth rates in nude mice was determined by repeated-measures ANOVA. Mann-Whitney U test was used for comparing means between two groups. *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/437/eaap9840/DC1

    Fig. S1. SQLE expression in NAFLD-HCC is controlled by transcription factors.

    Fig. S2. Transcription factors SREBP2 and MEIS1 bind to SQLE promoter region and activate SQLE gene expression.

    Fig. S3. SQLE promotes HCC cell growth.

    Fig. S4. SQLE in HCC cell lines promoted cell cycle progression at G1-S phase and inhibited apoptosis induction.

    Fig. S5. Sqle tg expression in mice induced apoptosis and the expression of proinflammatory cytokines and chemokines.

    Fig. S6. Cholesteryl ester and cholesterol concentrations are increased in NAFLD-HCC.

    Fig. S7. Oncogenic function of SQLE is dependent on the PTEN/PI3K/AKT/mTOR pathway.

    Fig. S8. SQLE silences PTEN expression through ROS-mediated DNA hypermethylation.

    Fig. S9. SQLE inhibitor terbinafine suppressed NAFLD-HCC growth in vitro and in vivo.

    Table S1. Epigenetic Chromatin Modification Enzymes PCR Array data.

    Table S2. Hypermethylated genes in LO2-SQLE cells.

    Table S3. Hypomethylated genes in LO2-SQLE cells.

    Table S4. Clinicopathological features of SQLE mRNA expression in our HCC cohort.

    Table S5. Clinicopathological features of SQLE mRNA expression in TCGA HCC cohort.

    Table S6. Primers used in this study.

    Table S7. Antibodies used in this study.

  • Supplementary Material for:

    Squalene epoxidase drives NAFLD-induced hepatocellular carcinoma and is a pharmaceutical target

    Dabin Liu, Chi Chun Wong, Li Fu, Huarong Chen, Liuyang Zhao, Chuangen Li, Yunfei Zhou, Yanquan Zhang, Weiqi Xu, Yidong Yang, Bin Wu, Gong Cheng, Paul Bo-San Lai, Nathalie Wong, Joseph J. Y. Sung, Jun Yu*

    *Corresponding author. Email: junyu{at}cuhk.edu.hk

    Published 18 April 2018, Sci. Transl. Med. 10, eaap9840 (2018)
    DOI: 10.1126/scitranslmed.aap9840

    This PDF file includes:

    • Fig. S1. SQLE expression in NAFLD-HCC is controlled by transcription factors.
    • Fig. S2. Transcription factors SREBP2 and MEIS1 bind to SQLE promoter region and activate SQLE gene expression.
    • Fig. S3. SQLE promotes HCC cell growth.
    • Fig. S4. SQLE in HCC cell lines promoted cell cycle progression at G1-S phase and inhibited apoptosis induction.
    • Fig. S5. Sqle tg expression in mice induced apoptosis and the expression of proinflammatory cytokines and chemokines.
    • Fig. S6. Cholesteryl ester and cholesterol concentrations are increased in NAFLD-HCC.
    • Fig. S7. Oncogenic function of SQLE is dependent on the PTEN/PI3K/AKT/mTOR pathway.
    • Fig. S8. SQLE silences PTEN expression through ROS-mediated DNA hypermethylation.
    • Fig. S9. SQLE inhibitor terbinafine suppressed NAFLD-HCC growth in vitro and in vivo.
    • Table S1. Epigenetic Chromatin Modification Enzymes PCR Array data.
    • Table S2. Hypermethylated genes in LO2-SQLE cells.
    • Table S3. Hypomethylated genes in LO2-SQLE cells.
    • Table S4. Clinicopathological features of SQLE mRNA expression in our HCC cohort.
    • Table S5. Clinicopathological features of SQLE mRNA expression in TCGA HCC cohort.
    • Table S6. Primers used in this study.
    • Table S7. Antibodies used in this study.

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