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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

Suppression of growth of oncogenic livers in Myc/xmrk double transgenic larvae by Pkm2 activation.10 μg/ml TEPP-46 was used to treat zebrafish larvae from 4 dpf for 96 hours and 2D liver size was measured by using ImageJ (1.49J) and cell proliferation on cryosections were examined by immune-staining of PCNA. (A,B) Changes of 2D liver size. Representative images from each group are shown in (A) and quantification of 2D liver size is presented in (B). (C,D) Immunostaining of PCNA for cell proliferation. Representative images in the liver area from each experimental group are shown in (A) and quantification of PCNA positive cells as percentage of liver cells is presented in (B). Group designations: X for xmrk, M for Myc, and T for TEPP-46. + and—indicate presence and absence respectively. All groups also received 30 μg/ml doxycycline in the experimental duration (4–8 dpf). Statistical significance was examined by unpaired student t test. *P<0.05; **P<0.01; ****P<0.0001.
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pone.0132319.g007: Suppression of growth of oncogenic livers in Myc/xmrk double transgenic larvae by Pkm2 activation.10 μg/ml TEPP-46 was used to treat zebrafish larvae from 4 dpf for 96 hours and 2D liver size was measured by using ImageJ (1.49J) and cell proliferation on cryosections were examined by immune-staining of PCNA. (A,B) Changes of 2D liver size. Representative images from each group are shown in (A) and quantification of 2D liver size is presented in (B). (C,D) Immunostaining of PCNA for cell proliferation. Representative images in the liver area from each experimental group are shown in (A) and quantification of PCNA positive cells as percentage of liver cells is presented in (B). Group designations: X for xmrk, M for Myc, and T for TEPP-46. + and—indicate presence and absence respectively. All groups also received 30 μg/ml doxycycline in the experimental duration (4–8 dpf). Statistical significance was examined by unpaired student t test. *P<0.05; **P<0.01; ****P<0.0001.

Mentions: It has been reported that PKM2 has two forms: an active tetrameric form and a less active dimeric form; tumor cells have predominantly the inactive dimeric form, which causes incomplete glycolysis and anabolism to promote tumor growth [33, 34]. Thus, increase of pyruvate kinase activity results in inhibition of tumor growth; this has been demonstrated by using PKM2 specific activator, TEPP-46, to stabilize the tetrameric configuration of PKM2 [33]. To demonstrate a similar mechanism in the zebrafish liver cancer model, TEPP-46 was used to treat all the three transgenic larvae together with doxycycline. As shown in Fig 7A and 7B, all three transgenic larvae showed significantly enlarged livers when doxycycline was administrated at 30 μg/ml, with the Myc/xmrk larvae showing the highest enlargement. When TEPP-46 was applied together with doxycycline, Myc/xmrk larvae showed the most significant reduction in liver size (P = 0.0026) compared with milder reduction in xmrk larvae (P = 0.024) and no significant change in Myc larvae. To further understand the mechanism of TEPP-46 on liver reduction, PCNA staining were performed. As shown in Fig 7C and 7D, significant down-regulation of cell proliferation in the liver occurred in the TEPP-46 treated Myc/xmrk and xmrk larvae, and again there was no significant effect on Myc larvae. These observations suggested that Pkm2 enzyme activity is indeed involved in liver carcinogenesis through regulating cell proliferation.


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)

Suppression of growth of oncogenic livers in Myc/xmrk double transgenic larvae by Pkm2 activation.10 μg/ml TEPP-46 was used to treat zebrafish larvae from 4 dpf for 96 hours and 2D liver size was measured by using ImageJ (1.49J) and cell proliferation on cryosections were examined by immune-staining of PCNA. (A,B) Changes of 2D liver size. Representative images from each group are shown in (A) and quantification of 2D liver size is presented in (B). (C,D) Immunostaining of PCNA for cell proliferation. Representative images in the liver area from each experimental group are shown in (A) and quantification of PCNA positive cells as percentage of liver cells is presented in (B). Group designations: X for xmrk, M for Myc, and T for TEPP-46. + and—indicate presence and absence respectively. All groups also received 30 μg/ml doxycycline in the experimental duration (4–8 dpf). Statistical significance was examined by unpaired student t test. *P<0.05; **P<0.01; ****P<0.0001.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132319.g007: Suppression of growth of oncogenic livers in Myc/xmrk double transgenic larvae by Pkm2 activation.10 μg/ml TEPP-46 was used to treat zebrafish larvae from 4 dpf for 96 hours and 2D liver size was measured by using ImageJ (1.49J) and cell proliferation on cryosections were examined by immune-staining of PCNA. (A,B) Changes of 2D liver size. Representative images from each group are shown in (A) and quantification of 2D liver size is presented in (B). (C,D) Immunostaining of PCNA for cell proliferation. Representative images in the liver area from each experimental group are shown in (A) and quantification of PCNA positive cells as percentage of liver cells is presented in (B). Group designations: X for xmrk, M for Myc, and T for TEPP-46. + and—indicate presence and absence respectively. All groups also received 30 μg/ml doxycycline in the experimental duration (4–8 dpf). Statistical significance was examined by unpaired student t test. *P<0.05; **P<0.01; ****P<0.0001.
Mentions: It has been reported that PKM2 has two forms: an active tetrameric form and a less active dimeric form; tumor cells have predominantly the inactive dimeric form, which causes incomplete glycolysis and anabolism to promote tumor growth [33, 34]. Thus, increase of pyruvate kinase activity results in inhibition of tumor growth; this has been demonstrated by using PKM2 specific activator, TEPP-46, to stabilize the tetrameric configuration of PKM2 [33]. To demonstrate a similar mechanism in the zebrafish liver cancer model, TEPP-46 was used to treat all the three transgenic larvae together with doxycycline. As shown in Fig 7A and 7B, all three transgenic larvae showed significantly enlarged livers when doxycycline was administrated at 30 μg/ml, with the Myc/xmrk larvae showing the highest enlargement. When TEPP-46 was applied together with doxycycline, Myc/xmrk larvae showed the most significant reduction in liver size (P = 0.0026) compared with milder reduction in xmrk larvae (P = 0.024) and no significant change in Myc larvae. To further understand the mechanism of TEPP-46 on liver reduction, PCNA staining were performed. As shown in Fig 7C and 7D, significant down-regulation of cell proliferation in the liver occurred in the TEPP-46 treated Myc/xmrk and xmrk larvae, and again there was no significant effect on Myc larvae. These observations suggested that Pkm2 enzyme activity is indeed involved in liver carcinogenesis through regulating cell proliferation.

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