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Synergistic Induction of Potential Warburg Effect in Zebrafish Hepatocellular Carcinoma by Co-Transgenic Expression of Myc and xmrk Oncogenes.

Li Z, Zheng W, Li H, Li C, Gong Z - PLoS ONE (2015)

Bottom Line: RNA-Seq analyses revealed distinct changes in many molecular pathways among the three types of liver tumors.In RT-qPCR analyses, the specific pkm2 isoformin Warburg effect was found to be highly enriched in the Myc/xmrk tumors but not in Myc or xmrk tumors, consistent with the observations in many human cancers with Warburg effect.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, 117543, Singapore, Singapore.

ABSTRACT
Previously we have generated inducible liver tumor models by transgenic expression of Myc or xmrk (activated EGFR 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 Myc/xmrk 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 Myc/xmrk tumors. Critical glycolytic genes including hk2, pkm and ldha were significantly up-regulated in Myc/xmrk tumors but not in either single oncogene-induced tumors, suggesting a potential Warburg effect. In RT-qPCR analyses, the specific pkm2 isoformin Warburg effect was found to be highly enriched in the Myc/xmrk tumors but not in Myc or xmrk tumors, consistent with the observations in many human cancers with Warburg effect. Moreover, the splicing factor genes (hnrnpa1, ptbp1a, ptbp1b and sfrs3b) responsible for generating the pkm isoform were also greatly up-regulated in the Myc/xmrk 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 Myc/xmrk zebrafish model suggests synergetic effect of EGFR and MYC in triggering Warburg effect in the HCC formation and may provide a promising in vivo model for Warburg effect.

No MeSH data available.


Related in: MedlinePlus

Expression of Warburg effect genes.(A) Expression of glycolytic genes in Myc, xmrk and Myc/xmrk tumors. (B) Expression of glycolytic genes splicing factors in Myc, xmrk and Myc/xmrk tumors. Asterisks indicate significantly changed genes (fold change>1.5, P<0.05). (C) Schematic comparison of genomic structure of human and zebrafish PKM1/pkm1and PKM2/pkm2 isoforms. The primers used for RT-qPCR are indicated by arrowheads. (D) RT-qPCR quantification of zebrafish pkm1 and pkm2expression in three tumor samples as compared with non-tumor controls. ***P<0.001; ****P<0.0001. (E) RT-qPCR quantification of pklr expression in three tumor samples as compared with non-tumor controls. ****P<0.0001.
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pone.0132319.g006: Expression of Warburg effect genes.(A) Expression of glycolytic genes in Myc, xmrk and Myc/xmrk tumors. (B) Expression of glycolytic genes splicing factors in Myc, xmrk and Myc/xmrk tumors. Asterisks indicate significantly changed genes (fold change>1.5, P<0.05). (C) Schematic comparison of genomic structure of human and zebrafish PKM1/pkm1and PKM2/pkm2 isoforms. The primers used for RT-qPCR are indicated by arrowheads. (D) RT-qPCR quantification of zebrafish pkm1 and pkm2expression in three tumor samples as compared with non-tumor controls. ***P<0.001; ****P<0.0001. (E) RT-qPCR quantification of pklr expression in three tumor samples as compared with non-tumor controls. ****P<0.0001.

Mentions: As indicated in Fig 5, several important metabolomic pathways were up-regulated inMyc/xmrk tumors. In particular, Acetyl-CoA is an important mediator between glucose, fatty acid and amino acid metabolism and TCA cycle, and these up-regulated metabolic pathways indicated a high requirement of glucose, fatty acid and amino acid metabolism. However, the fatty acid and amino acid metabolism were down-regulated in the Myc/xmrk tumors, suggesting a major dependence of energy production through glucose metabolism. Thus, we envisage an existence of Warburg effect in the Myc/xmrk tumors, which describes the phenomena that many cancer cells produce energy by a high rate of glycolysis followed by lactic acid fermentation even in the presence of sufficient oxygen [30]. To confirm this, we examined expression of genes for critical enzymes in glycolysis and observed significant up-regulation of glycolytic genes includinghk2, pkm and ldha in the Myc/xmrk tumors, whereas none of them showed significantly changed in either Myc or xmrk tumors (Fig 6A), thus strongly suggesting up-regulation of the glycolysis process or Warburg effect uniquely in the Myc/xmrk tumors. Moreover, splicing factors for the glycolytic genes including hnrnpa, ptbp1a, ptbp1b and sfrs3b were all further up-regulated in the Myc/xmrk tumors compared with the xmrk tumor, whereas no significant change was observed in theMyctumors (Fig 6B), indicating potentially different splicing activities in these different types of tumors.


Synergistic Induction of Potential Warburg Effect in Zebrafish Hepatocellular Carcinoma by Co-Transgenic Expression of Myc and xmrk Oncogenes.

Li Z, Zheng W, Li H, Li C, Gong Z - PLoS ONE (2015)

Expression of Warburg effect genes.(A) Expression of glycolytic genes in Myc, xmrk and Myc/xmrk tumors. (B) Expression of glycolytic genes splicing factors in Myc, xmrk and Myc/xmrk tumors. Asterisks indicate significantly changed genes (fold change>1.5, P<0.05). (C) Schematic comparison of genomic structure of human and zebrafish PKM1/pkm1and PKM2/pkm2 isoforms. The primers used for RT-qPCR are indicated by arrowheads. (D) RT-qPCR quantification of zebrafish pkm1 and pkm2expression in three tumor samples as compared with non-tumor controls. ***P<0.001; ****P<0.0001. (E) RT-qPCR quantification of pklr expression in three tumor samples as compared with non-tumor controls. ****P<0.0001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492623&req=5

pone.0132319.g006: Expression of Warburg effect genes.(A) Expression of glycolytic genes in Myc, xmrk and Myc/xmrk tumors. (B) Expression of glycolytic genes splicing factors in Myc, xmrk and Myc/xmrk tumors. Asterisks indicate significantly changed genes (fold change>1.5, P<0.05). (C) Schematic comparison of genomic structure of human and zebrafish PKM1/pkm1and PKM2/pkm2 isoforms. The primers used for RT-qPCR are indicated by arrowheads. (D) RT-qPCR quantification of zebrafish pkm1 and pkm2expression in three tumor samples as compared with non-tumor controls. ***P<0.001; ****P<0.0001. (E) RT-qPCR quantification of pklr expression in three tumor samples as compared with non-tumor controls. ****P<0.0001.
Mentions: As indicated in Fig 5, several important metabolomic pathways were up-regulated inMyc/xmrk tumors. In particular, Acetyl-CoA is an important mediator between glucose, fatty acid and amino acid metabolism and TCA cycle, and these up-regulated metabolic pathways indicated a high requirement of glucose, fatty acid and amino acid metabolism. However, the fatty acid and amino acid metabolism were down-regulated in the Myc/xmrk tumors, suggesting a major dependence of energy production through glucose metabolism. Thus, we envisage an existence of Warburg effect in the Myc/xmrk tumors, which describes the phenomena that many cancer cells produce energy by a high rate of glycolysis followed by lactic acid fermentation even in the presence of sufficient oxygen [30]. To confirm this, we examined expression of genes for critical enzymes in glycolysis and observed significant up-regulation of glycolytic genes includinghk2, pkm and ldha in the Myc/xmrk tumors, whereas none of them showed significantly changed in either Myc or xmrk tumors (Fig 6A), thus strongly suggesting up-regulation of the glycolysis process or Warburg effect uniquely in the Myc/xmrk tumors. Moreover, splicing factors for the glycolytic genes including hnrnpa, ptbp1a, ptbp1b and sfrs3b were all further up-regulated in the Myc/xmrk tumors compared with the xmrk tumor, whereas no significant change was observed in theMyctumors (Fig 6B), indicating potentially different splicing activities in these different types of tumors.

Bottom Line: RNA-Seq analyses revealed distinct changes in many molecular pathways among the three types of liver tumors.In RT-qPCR analyses, the specific pkm2 isoformin Warburg effect was found to be highly enriched in the Myc/xmrk tumors but not in Myc or xmrk tumors, consistent with the observations in many human cancers with Warburg effect.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, 117543, Singapore, Singapore.

ABSTRACT
Previously we have generated inducible liver tumor models by transgenic expression of Myc or xmrk (activated EGFR 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 Myc/xmrk 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 Myc/xmrk tumors. Critical glycolytic genes including hk2, pkm and ldha were significantly up-regulated in Myc/xmrk tumors but not in either single oncogene-induced tumors, suggesting a potential Warburg effect. In RT-qPCR analyses, the specific pkm2 isoformin Warburg effect was found to be highly enriched in the Myc/xmrk tumors but not in Myc or xmrk tumors, consistent with the observations in many human cancers with Warburg effect. Moreover, the splicing factor genes (hnrnpa1, ptbp1a, ptbp1b and sfrs3b) responsible for generating the pkm isoform were also greatly up-regulated in the Myc/xmrk 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 Myc/xmrk zebrafish model suggests synergetic effect of EGFR and MYC in triggering Warburg effect in the HCC formation and may provide a promising in vivo model for Warburg effect.

No MeSH data available.


Related in: MedlinePlus