Synergistic Induction of Potential Warburg Effect in Zebrafish Hepatocellular Carcinoma by Co-Transgenic Expression of <i>Myc</i> and <i>xmrk</i> Oncogenes

<div><p>Previously we have generated inducible liver tumor models by transgenic expression of <i>Myc</i> or <i>xmrk</i> (activated <i>EGFR</i> homolog) oncogenes in zebrafish. To investigate the interaction of the two oncogenes, we crossed the two transgenic lines and observed more severe and faster hepatocarcinogenesis in <i>Myc</i>/<i>xmrk</i> double transgenic zebrafish than either single transgenic fish. RNA-Seq analyses revealed distinct changes in many molecular pathways among the three types of liver tumors. In particular, we found dramatic alteration of cancer metabolism based on the uniquely enriched pathways in the <i>Myc/xmrk</i> tumors. Critical glycolytic genes including <i>hk2</i>, <i>pkm</i> and <i>ldha</i> were significantly up-regulated in <i>Myc/xmrk</i> tumors but not in either single oncogene-induced tumors, suggesting a potential Warburg effect. In RT-qPCR analyses, the specific <i>pkm2</i> isoformin Warburg effect was found to be highly enriched in the <i>Myc/xmrk</i> tumors but not in <i>Myc</i> or <i>xmrk</i> tumors, consistent with the observations in many human cancers with Warburg effect. Moreover, the splicing factor genes (<i>hnrnpa1</i>, <i>ptbp1a</i>, <i>ptbp1b and sfrs3b</i>) responsible for generating the <i>pkm</i> isoform were also greatly up-regulated in the <i>Myc/xmrk</i> tumors. As Pkm2 isoform is generally inactive and causes incomplete glycolysis to favor anabolism and tumor growth, by treatment with a Pkm2-specific activator, TEPP-46, we further demonstrated that activation of Pkm2 suppressed the growth of oncogenic liver as well as proliferation of liver cells. Collectively, our <i>Myc/xmrk</i> zebrafish model suggests synergetic effect of EGFR and MYC in triggering Warburg effect in the HCC formation and may provide a promising <i>in vivo</i> model for Warburg effect.</p></div

RNA-Seq analyses of <i>Myc</i>-, <i>xmrk</i>-, <i>Myc/xmrk</i>-induced liver tumors.

<p>(A) Hierarchical clustering of the eight RNA-Seq samples.(B) Venn diagram of up- and down-regulated transcripts in the three liver tumors. (C) Venn diagram of up-and down-regulated canonical pathways in the three liver tumors. (D) Venn diagram of pathways with opposite directions between <i>Myc</i>- and <i>xmrk</i>-induced liver tumors.</p

Uniquely up- and down-regulated biological process (BP) and KEGG pathways in <i>Myc/xmrk</i> liver tumors.

<p>Uniquely up- or down-regulated genes in the <i>Myc/xmrk</i> tumors (1,114 and 359 genes respectively as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132319#pone.0132319.g002" target="_blank">Fig 2B</a>) were input into the DAVID online software and top BPs and KEGG pathways are shown. (A) Top significantly up-regulated BPs. (B) Significantly up-regulated KEGG pathway with P<0.05 and Benjamini value<0.05 cutoff. (C) Significantly down-regulated KEGG pathway with P<0.05 and Benjamini value<0.05 cutoff. Negative log P-value was plotted against different processes/pathways.</p

Expression of Warburg effect genes.

<p>(A) Expression of glycolytic genes in <i>Myc</i>, <i>xmrk</i> and <i>Myc/xmrk</i> tumors. (B) Expression of glycolytic genes splicing factors in <i>Myc</i>, <i>xmrk</i> and <i>Myc/xmrk</i> tumors. Asterisks indicate significantly changed genes (fold change>1.5, P<0.05). (C) Schematic comparison of genomic structure of human and zebrafish <i>PKM1/pkm1</i>and <i>PKM2/pkm2</i> isoforms. The primers used for RT-qPCR are indicated by arrowheads. (D) RT-qPCR quantification of zebrafish <i>pkm1</i> and <i>pkm2</i>expression in three tumor samples as compared with non-tumor controls. ***P<0.001; ****P<0.0001. (E) RT-qPCR quantification of <i>pklr</i> expression in three tumor samples as compared with non-tumor controls. ****P<0.0001.</p

Counteractive effects of pathways oppositely regulated by <i>Myc</i> and <i>xmrk</i>.

<p>All but one pathway which were oppositely regulated by <i>Myc</i> and <i>xmrk</i> were counterbalanced and did not show any significant changes in the <i>Myc</i>/<i>xmrk</i> transgenic liver cancer. Red colors indicate up-regulation while green color down-regulation. FDR values are shown in different color gradients as indicated.</p

Synergistic effect of <i>Myc</i> and <i>xmrk</i> oncogenes in transgenic zebrafish survival and liver tumorigenesis.

<p>(A,B) Survival curve (A) and gross morphology (B) of oncogene transgenic zebrafish following doxycycline induction at the juvenile stage (starting from 21 dpf). (C) Survival curve of oncogene transgenic zebrafish following doxycycline induction at the adult stage (starting from 3.5 mpf). (D) Gross observation of liver phenotype (left) and histological sections of livers stained by hematoxylin and eosin dyes (right). Abbreviations: X,<i>xmrk;</i> M,<i>Myc;</i> D, doxycycline treatment.</p

Synergetic effects of pathways co-regulated by <i>Myc</i> and <i>xmrk</i>.

<p>44 canonical pathways were identified to be up-regulated in both <i>Myc</i>- and <i>xmrk</i>-induced zebrafish liver cancer. 32 out of them showed more significant up-regulation in the <i>Myc</i>/<i>xmrk</i> transgenic liver cancer. FDR values are shown in different color gradient as indicated.</p

Comparison of transcriptomic profiles between TCDD and control groups.

<p>(A) Distribution of transcript entries and total transcript counts over different tag abundance categories in liver of zebrafish. The percentages of total transcript counts and number of different transcript entries per category are plotted on a log scale (base10). (B) Relationship between the hepatic transcriptome changing range and its expression level in zebrafish after TCDD treatment. The base of log value is 2.</p

RNA-Sequencing Analysis of TCDD-Induced Responses in Zebrafish Liver Reveals High Relatedness to <i>In Vivo</i> Mammalian Models and Conserved Biological Pathways

<div><p>TCDD is one of the most persistent environmental toxicants in biological systems and its effect through aryl hydrocarbon receptor (AhR) has been well characterized. However, the information on TCDD-induced toxicity in other molecular pathways is rather limited. To fully understand molecular toxicity of TCDD in an in vivo animal model, adult zebrafish were exposed to TCDD at 10 nM for 96 h and the livers were sampled for RNA-sequencing based transcriptomic profiling. A total of 1,058 differently expressed genes were identified based on fold-change>2 and TPM (transcripts per million) >10. Among the top 20 up-regulated genes, 10 novel responsive genes were identified and verified by RT-qPCR analysis on independent samples. Transcriptomic analysis indicated several deregulated pathways associated with cell cycle, endocrine disruptors, signal transduction and immune systems. Comparative analyses of TCDD-induced transcriptomic changes between fish and mammalian models revealed that proteomic pathway is consistently up-regulated while calcium signaling pathway and several immune-related pathways are generally down-regulated. Finally, our study also suggested that zebrafish model showed greater similarity to <i>in vivo</i> mammalian models than <i>in vitro</i> models. Our study indicated that the zebrafish is a valuable in vivo model in toxicogenomic analyses for understanding molecular toxicity of environmental toxicants relevant to human health. The expression profiles associated with TCDD could be useful for monitoring environmental dioxin and dioxin-like contamination.</p></div

Comparative analyses of zebrafish and mammalian transcriptomic data from TCDD treatments.

<p>(A) Correlation of hepatic transcriptome changes in the zebrafish with the mammalian in vivo and in vitro models by GSEA analysis. –Log2FDR = 2, that means FDR = 0.25. (B) Comparison of the pathways in zebrafish and the mammalian models by GSEA analysis. The heat map includes the significant pathways in zebrafish and mammalian models treated by TCDD, the criteria of zebrafish pathways is FDR<0.25.</p