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Identification of Chemical Inhibitors of β-Catenin-Driven Liver Tumorigenesis in Zebrafish.

Evason KJ, Francisco MT, Juric V, Balakrishnan S, Lopez Pazmino Mdel P, Gordan JD, Kakar S, Spitsbergen J, Goga A, Stainier DY - PLoS Genet. (2015)

Bottom Line: As expected for a malignant process, transgenic zebrafish showed significantly decreased mean adult survival compared to non-transgenic control siblings.We further found that activated β-catenin was associated with JNK pathway hyperactivation in zebrafish and in human HCC.The β-catenin-specific growth-inhibitory effect of targeting JNK was conserved in human liver cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of California, San Francisco, San Francisco, California, United States of America; Department of Biochemistry and Biophysics, Programs in Developmental and Stem Cell Biology, Genetics and Human Genetics, Diabetes Center, Institute for Regeneration Medicine and the Liver Center, University of California, San Francisco, San Francisco, California, United States of America.

ABSTRACT
Hepatocellular carcinoma (HCC) is one of the most lethal human cancers. The search for targeted treatments has been hampered by the lack of relevant animal models for the genetically diverse subsets of HCC, including the 20-40% of HCCs that are defined by activating mutations in the gene encoding β-catenin. To address this chemotherapeutic challenge, we created and characterized transgenic zebrafish expressing hepatocyte-specific activated β-catenin. By 2 months post fertilization (mpf), 33% of transgenic zebrafish developed HCC in their livers, and 78% and 80% of transgenic zebrafish showed HCC at 6 and 12 mpf, respectively. As expected for a malignant process, transgenic zebrafish showed significantly decreased mean adult survival compared to non-transgenic control siblings. Using this novel transgenic model, we screened for druggable pathways that mediate β-catenin-induced liver growth and identified two c-Jun N-terminal kinase (JNK) inhibitors and two antidepressants (one tricyclic antidepressant, amitriptyline, and one selective serotonin reuptake inhibitor) that suppressed this phenotype. We further found that activated β-catenin was associated with JNK pathway hyperactivation in zebrafish and in human HCC. In zebrafish larvae, JNK inhibition decreased liver size specifically in the presence of activated β-catenin. The β-catenin-specific growth-inhibitory effect of targeting JNK was conserved in human liver cancer cells. Our other class of hits, antidepressants, has been used in patient treatment for decades, raising the exciting possibility that these drugs could potentially be repurposed for cancer treatment. In support of this proposal, we found that amitriptyline decreased tumor burden in a mouse HCC model. Our studies implicate JNK inhibitors and antidepressants as potential therapeutics for β-catenin-induced liver tumors.

No MeSH data available.


Related in: MedlinePlus

JNK inhibitors suppress larval liver enlargement caused by activated β-catenin.(A) Brightfield images of control sibling and transgenic 6-day-old fixed larvae, treated with DMSO or JNK inhibitors. Livers are outlined. Scale bar, 100 μm. (B-C) Graphs showing average liver size ± SEM of 6-day-old control sibling and transgenic larvae treated for 3 days with SP600125 (B) or EMD 420123 (C) at the indicated dosages. N values are shown above the x-axis. Asterisks indicate p-values for 2-way ANOVA comparing drug-treated zebrafish to DMSO-treated siblings with the same genotype: *, p<0.05; ***, p<0.001.
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pgen.1005305.g004: JNK inhibitors suppress larval liver enlargement caused by activated β-catenin.(A) Brightfield images of control sibling and transgenic 6-day-old fixed larvae, treated with DMSO or JNK inhibitors. Livers are outlined. Scale bar, 100 μm. (B-C) Graphs showing average liver size ± SEM of 6-day-old control sibling and transgenic larvae treated for 3 days with SP600125 (B) or EMD 420123 (C) at the indicated dosages. N values are shown above the x-axis. Asterisks indicate p-values for 2-way ANOVA comparing drug-treated zebrafish to DMSO-treated siblings with the same genotype: *, p<0.05; ***, p<0.001.

Mentions: As many anti-cancer agents are protein kinase inhibitors, we next applied our screening platform to a commercially available kinase inhibitor library. We tested 160 compounds and identified five drugs that decreased liver size in Tg(fabp10a:pt-β-cat) zebrafish without causing obvious toxicity (S5 Table). We selected SP600125 and EMD 420123 for additional follow-up studies because they were the only 2 of the 5 hits among the kinase inhibitors with a shared mechanism of action. SP600125 is a well-established JNK inhibitor[33]. EMD 420123 also inhibits JNK, albeit with a substantially higher IC50 in vitro[33]. At doses greater than or equal to 10 μM, both drugs caused edema and/or death. All larvae (34/34 and 27/27) treated with 10 μM or 50 μM EMD 420123 died. Five out of 31 and 7/31 larvae treated with 10 μM SP600125 died or showed edema, respectively; 2/21 and 17/21 larvae treated with 50 μM SP600125 died or showed edema; and 2/10 and 8/10 larvae treated with 100 μM SP600125 died or showed edema. At lower doses, both SP600125 and EMD 420123 caused a dose-dependent decrease in liver size in Tg(fabp10a:pt-β-cat) larvae (Fig 4A–4C). Although EMD 420123 also decreased liver size in non-transgenic animals, the magnitude of this effect was less substantial (20–40% decrease at 2–5 μM) than the effect on zebrafish expressing activated β-catenin (30–60% decrease at 2–5 μM). Furthermore, at doses up to 5 μM, SP600125 did not affect liver size in non-transgenic animals, suggesting that the decrease in liver size we observed with this compound was related to specific effects in the context of activated β-catenin and not due to non-specific toxicity.


Identification of Chemical Inhibitors of β-Catenin-Driven Liver Tumorigenesis in Zebrafish.

Evason KJ, Francisco MT, Juric V, Balakrishnan S, Lopez Pazmino Mdel P, Gordan JD, Kakar S, Spitsbergen J, Goga A, Stainier DY - PLoS Genet. (2015)

JNK inhibitors suppress larval liver enlargement caused by activated β-catenin.(A) Brightfield images of control sibling and transgenic 6-day-old fixed larvae, treated with DMSO or JNK inhibitors. Livers are outlined. Scale bar, 100 μm. (B-C) Graphs showing average liver size ± SEM of 6-day-old control sibling and transgenic larvae treated for 3 days with SP600125 (B) or EMD 420123 (C) at the indicated dosages. N values are shown above the x-axis. Asterisks indicate p-values for 2-way ANOVA comparing drug-treated zebrafish to DMSO-treated siblings with the same genotype: *, p<0.05; ***, p<0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4489858&req=5

pgen.1005305.g004: JNK inhibitors suppress larval liver enlargement caused by activated β-catenin.(A) Brightfield images of control sibling and transgenic 6-day-old fixed larvae, treated with DMSO or JNK inhibitors. Livers are outlined. Scale bar, 100 μm. (B-C) Graphs showing average liver size ± SEM of 6-day-old control sibling and transgenic larvae treated for 3 days with SP600125 (B) or EMD 420123 (C) at the indicated dosages. N values are shown above the x-axis. Asterisks indicate p-values for 2-way ANOVA comparing drug-treated zebrafish to DMSO-treated siblings with the same genotype: *, p<0.05; ***, p<0.001.
Mentions: As many anti-cancer agents are protein kinase inhibitors, we next applied our screening platform to a commercially available kinase inhibitor library. We tested 160 compounds and identified five drugs that decreased liver size in Tg(fabp10a:pt-β-cat) zebrafish without causing obvious toxicity (S5 Table). We selected SP600125 and EMD 420123 for additional follow-up studies because they were the only 2 of the 5 hits among the kinase inhibitors with a shared mechanism of action. SP600125 is a well-established JNK inhibitor[33]. EMD 420123 also inhibits JNK, albeit with a substantially higher IC50 in vitro[33]. At doses greater than or equal to 10 μM, both drugs caused edema and/or death. All larvae (34/34 and 27/27) treated with 10 μM or 50 μM EMD 420123 died. Five out of 31 and 7/31 larvae treated with 10 μM SP600125 died or showed edema, respectively; 2/21 and 17/21 larvae treated with 50 μM SP600125 died or showed edema; and 2/10 and 8/10 larvae treated with 100 μM SP600125 died or showed edema. At lower doses, both SP600125 and EMD 420123 caused a dose-dependent decrease in liver size in Tg(fabp10a:pt-β-cat) larvae (Fig 4A–4C). Although EMD 420123 also decreased liver size in non-transgenic animals, the magnitude of this effect was less substantial (20–40% decrease at 2–5 μM) than the effect on zebrafish expressing activated β-catenin (30–60% decrease at 2–5 μM). Furthermore, at doses up to 5 μM, SP600125 did not affect liver size in non-transgenic animals, suggesting that the decrease in liver size we observed with this compound was related to specific effects in the context of activated β-catenin and not due to non-specific toxicity.

Bottom Line: As expected for a malignant process, transgenic zebrafish showed significantly decreased mean adult survival compared to non-transgenic control siblings.We further found that activated β-catenin was associated with JNK pathway hyperactivation in zebrafish and in human HCC.The β-catenin-specific growth-inhibitory effect of targeting JNK was conserved in human liver cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of California, San Francisco, San Francisco, California, United States of America; Department of Biochemistry and Biophysics, Programs in Developmental and Stem Cell Biology, Genetics and Human Genetics, Diabetes Center, Institute for Regeneration Medicine and the Liver Center, University of California, San Francisco, San Francisco, California, United States of America.

ABSTRACT
Hepatocellular carcinoma (HCC) is one of the most lethal human cancers. The search for targeted treatments has been hampered by the lack of relevant animal models for the genetically diverse subsets of HCC, including the 20-40% of HCCs that are defined by activating mutations in the gene encoding β-catenin. To address this chemotherapeutic challenge, we created and characterized transgenic zebrafish expressing hepatocyte-specific activated β-catenin. By 2 months post fertilization (mpf), 33% of transgenic zebrafish developed HCC in their livers, and 78% and 80% of transgenic zebrafish showed HCC at 6 and 12 mpf, respectively. As expected for a malignant process, transgenic zebrafish showed significantly decreased mean adult survival compared to non-transgenic control siblings. Using this novel transgenic model, we screened for druggable pathways that mediate β-catenin-induced liver growth and identified two c-Jun N-terminal kinase (JNK) inhibitors and two antidepressants (one tricyclic antidepressant, amitriptyline, and one selective serotonin reuptake inhibitor) that suppressed this phenotype. We further found that activated β-catenin was associated with JNK pathway hyperactivation in zebrafish and in human HCC. In zebrafish larvae, JNK inhibition decreased liver size specifically in the presence of activated β-catenin. The β-catenin-specific growth-inhibitory effect of targeting JNK was conserved in human liver cancer cells. Our other class of hits, antidepressants, has been used in patient treatment for decades, raising the exciting possibility that these drugs could potentially be repurposed for cancer treatment. In support of this proposal, we found that amitriptyline decreased tumor burden in a mouse HCC model. Our studies implicate JNK inhibitors and antidepressants as potential therapeutics for β-catenin-induced liver tumors.

No MeSH data available.


Related in: MedlinePlus