Evason KJ et al. (2015), Identification of Chemical Inhibitors of β-Cate...

XB-ART-53308

PLoS Genet 2015 Jul 01;117:e1005305. doi: 10.1371/journal.pgen.1005305.

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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 .

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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.

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Species referenced: Xenopus laevis
Genes referenced: ctnnb1 emd hccs jun mapk8 mhc2-dab

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	Fig 1. Hepatocyte-specific expression of activated β-catenin results in liver enlargement, hepatocellular carcinoma (HCC), and decreased survival in adult zebrafish.(A-C) Control 4-month-old zebrafish showing the liver (L, outlined) positioned in the body cavity near the intestine (i) and swim bladder (sb)(A). Sections show an orderly arrangement of hepatocytes (B-C). (D-F) Transgenic 4-month-old zebrafish showing an enlarged liver (D) with disorganized architecture (E) and atypical cells (arrows, F). (G-H) Transgenic 6-month-old (G) or 4-month-old (H) zebrafish and human HCC showing architectural disruption with scattered pseudoglands (arrows, G) and evidence of intracellular lipid accumulation (arrows, H). Hematoxylin and eosin stains; scale bars, 1 mm (A, D), 100 μm (B, E), 25 μm (C, F), and 20 μm (G, H). (I) Graph showing average liver size normalized to total body mass, ± standard error of the mean (SEM). Asterisks indicate p-values for ANOVA comparing transgenic zebrafish (N = 56) to control siblings (N = 51) at the same time point: , p<0.05; **, p<0.001. (J) Livers of transgenic zebrafish (Tg, N = 49) and control siblings (C, N = 37) were examined microscopically, and architectural and cytological changes were scored. HCC was significantly more common in transgenic zebrafish than in controls (p<0.001, Fisher’s exact test). (K) Kaplan-Meier survival curves comparing adult survival of transgenic zebrafish (N = 51) and control siblings (N = 85); p<0.001, logrank test.
	Fig 2. Cross-species comparison of Tg(fabp10a:pt-β-cat) zebrafish and human HCC.(A-B) Analyses of Tg(fabp10a:pt-β-cat) zebrafish RNA-seq data and publicly available human microarray data based on a subset of 283 orthologous gene pairs that are significantly dys-regulated in human HCC samples when compared to adjacent non-tumor human liver samples. (A) Dendrogram illustrating hierarchical clustering of merged human and zebrafish samples, showing that 5 of 7 transgenic zebrafish livers cluster with human HCC and 4 of 5 non-transgenic zebrafish livers cluster with non-tumor human liver. (B) Principal component analysis of Tg(fabp10a:pt-β-cat) zebrafish livers, non-transgenic control livers, human HCC, and non-tumor human liver. Ellipses indicate the 95% confidence intervals of the first two principal components for the respective groups. (C) Heatmaps of human and zebrafish orthologs that are significantly dys-regulated in the same direction in both human HCC samples and Tg(fabp10a:pt-β-cat) animals when compared to their non-tumor counterparts (FDR < 0.05).
	Fig 3. Activated β-catenin causes larval liver enlargement and increased hepatocyte proliferation.(A) Brightfield images of control sibling and transgenic 6-day-old fixed larvae. Livers are outlined. Scale bars, 100 μm. (B) Graph showing average liver size ± SEM of 6-day-old larvae from three different transgenic lines (s985, s986, s987) compared to control siblings (C). N values are shown above the x-axis. Asterisks indicate p-values for 2-way ANOVA comparing transgenic zebrafish to control siblings in the same experiment: , p<0.01; , p<0.001. (C) Immunofluorescence images of 6-day-old control and transgenic larvae, highlighting hepatocyte cell membranes (Tg(fabp10a:rasGFP)) and proliferating cells (EdU). Scale bars, 20 μm. Inset photos are 4X magnifications. (D-E) Graphs showing average cell size ± SEM (D) and percent of hepatocytes that were EdU positive (E). N values, which are the same for both graphs, appear above the x-axis in D; samples were compared using the Student’s t-test. , p<0.05.
	Fig 4. 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.
	Fig 5. Activated β-catenin is associated with JNK pathway activation.(A) Representative whole-mount immunofluorescence images and (B) quantification of 5-day-old control sibling and transgenic larvae treated for 2 days with 0.5% DMSO alone, 5 μM SP600125, or 5 μM EMD 420123 and stained with antibodies against phospho-c-Jun (left panels) and TO-PRO nuclear stain (right panels). Livers are outlined in white. Scale bar, 20 μm. N values are shown above the x-axis. Drug-treated zebrafish were compared to DMSO-treated siblings with the same genotype using 2-way ANOVA. **, p<0.001. (C) Representative photographs of zebrafish control and Tg(fabp10a:pt-β-cat) livers (left) and human HCC without (middle) and with (right) activated β-catenin, stained with anti-phospho-c-Jun antibodies. For zebrafish, 6 out of 8 (75%) Tg(fabp10a:pt-β-cat) livers with HCC contained one or more foci with moderate or high nuclear phospho-c-Jun staining (phospho-c-Jun positive), while all non-transgenic control livers (N = 7) without activated β-catenin showed absent or low staining (phospho-c-Jun negative). For human HCC, the number of cases exhibiting each staining pattern and the total number of cases with a given β-catenin activation status are shown at the bottom right of each picture. Images counterstained with hematoxylin. Scale bars, 20 μm. (D) Normalized jun mRNA expression in control sibling and transgenic adult zebrafish livers. Three technical replicates were performed for each sample. , p<0.05, Mann-Whitney test. N values are shown above the x-axis. (E) Normalized JUN mRNA expression for human HCC with low GLUL and high GLUL expression. **, p<0.001, Mann-Whitney test. (F) Graph showing dose of JNK inhibitor CC401 at which cell viability of human cancer cell lines was decreased by 50% (GI50), ± SEM. Number of replicates for each cell line is shown above the x-axis. , p<0.05, unpaired t-test.
	Fig 6. Antidepressants decrease β-catenin-induced liver enlargement and tumorigenesis.(A-B) Graphs showing mean liver size ± SEM of 6-day-old control sibling and transgenic zebrafish larvae treated for 3 days with amitriptyline (A) or paroxetine (B) 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<0.01; , p<0.001. (C) Graph showing mean liver size ± SEM of 6-day-old control sibling and transgenic zebrafish larvae treated for 3 days with 0.5% DMSO (-), 20 μM amitriptyline (AMI), 2 μM SP600125 (SP), or both drugs combined (AMI+SP). N values are shown above the x-axis. Asterisks indicate p-values for 2-way ANOVA comparing each group of transgenic zebrafish to AMI+SP group: , p<0.01; ***, p<0.001. N.S., no significant difference between groups of non-transgenic zebrafish (2-way ANOVA). (D) Representative images of control, non-hydrodynamically transfected (non-HDT) mouse livers (left) and mouse liver tumors induced by hydrodynamic transfection of activated β-catenin and Met (Met/β-cat HDT). Mice were treated with saccharine alone (vehicle only, top row) or amitriptyline plus saccharine (bottom row). Scale bars, 1 cm. (E) Graph showing mean liver-to-body mass ratios ± SEM for non-HDT and HDT-Met/β-cat mice treated with saccharine alone (vehicle) or amitriptyline plus saccharine (+Ami). P values calculated with Mann-Whitney test. N values are shown above the x-axis. (F) Representative hematoxylin-and-eosin-stained, Ki-67-labeled, and TUNEL-labeled images from vehicle- and amitriptyline-treated mice. Ki-67 and TUNEL stainings were performed using 3, 3'-diaminobenzidine (DAB) substrate, so positive-staining cells are brown, and hematoxylin counterstain to highlight nuclei and other basophilic structures in blue. (G-H) Graphs showing mean ± SEM of Ki-67-positive (G) or TUNEL-positive (H) cells per high-power field. P values calculated with Mann-Whitney test. N values are shown above the x-axis.

References [+] :

Ashburner, Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. 2000, Pubmed

	Fig 1. Hepatocyte-specific expression of activated β-catenin results in liver enlargement, hepatocellular carcinoma (HCC), and decreased survival in adult zebrafish.(A-C) Control 4-month-old zebrafish showing the liver (L, outlined) positioned in the body cavity near the intestine (i) and swim bladder (sb)(A). Sections show an orderly arrangement of hepatocytes (B-C). (D-F) Transgenic 4-month-old zebrafish showing an enlarged liver (D) with disorganized architecture (E) and atypical cells (arrows, F). (G-H) Transgenic 6-month-old (G) or 4-month-old (H) zebrafish and human HCC showing architectural disruption with scattered pseudoglands (arrows, G) and evidence of intracellular lipid accumulation (arrows, H). Hematoxylin and eosin stains; scale bars, 1 mm (A, D), 100 μm (B, E), 25 μm (C, F), and 20 μm (G, H). (I) Graph showing average liver size normalized to total body mass, ± standard error of the mean (SEM). Asterisks indicate p-values for ANOVA comparing transgenic zebrafish (N = 56) to control siblings (N = 51) at the same time point: , p<0.05; **, p<0.001. (J) Livers of transgenic zebrafish (Tg, N = 49) and control siblings (C, N = 37) were examined microscopically, and architectural and cytological changes were scored. HCC was significantly more common in transgenic zebrafish than in controls (p<0.001, Fisher’s exact test). (K) Kaplan-Meier survival curves comparing adult survival of transgenic zebrafish (N = 51) and control siblings (N = 85); p<0.001, logrank test.
	Fig 2. Cross-species comparison of Tg(fabp10a:pt-β-cat) zebrafish and human HCC.(A-B) Analyses of Tg(fabp10a:pt-β-cat) zebrafish RNA-seq data and publicly available human microarray data based on a subset of 283 orthologous gene pairs that are significantly dys-regulated in human HCC samples when compared to adjacent non-tumor human liver samples. (A) Dendrogram illustrating hierarchical clustering of merged human and zebrafish samples, showing that 5 of 7 transgenic zebrafish livers cluster with human HCC and 4 of 5 non-transgenic zebrafish livers cluster with non-tumor human liver. (B) Principal component analysis of Tg(fabp10a:pt-β-cat) zebrafish livers, non-transgenic control livers, human HCC, and non-tumor human liver. Ellipses indicate the 95% confidence intervals of the first two principal components for the respective groups. (C) Heatmaps of human and zebrafish orthologs that are significantly dys-regulated in the same direction in both human HCC samples and Tg(fabp10a:pt-β-cat) animals when compared to their non-tumor counterparts (FDR < 0.05).
	Fig 3. Activated β-catenin causes larval liver enlargement and increased hepatocyte proliferation.(A) Brightfield images of control sibling and transgenic 6-day-old fixed larvae. Livers are outlined. Scale bars, 100 μm. (B) Graph showing average liver size ± SEM of 6-day-old larvae from three different transgenic lines (s985, s986, s987) compared to control siblings (C). N values are shown above the x-axis. Asterisks indicate p-values for 2-way ANOVA comparing transgenic zebrafish to control siblings in the same experiment: , p<0.01; , p<0.001. (C) Immunofluorescence images of 6-day-old control and transgenic larvae, highlighting hepatocyte cell membranes (Tg(fabp10a:rasGFP)) and proliferating cells (EdU). Scale bars, 20 μm. Inset photos are 4X magnifications. (D-E) Graphs showing average cell size ± SEM (D) and percent of hepatocytes that were EdU positive (E). N values, which are the same for both graphs, appear above the x-axis in D; samples were compared using the Student’s t-test. , p<0.05.
	Fig 4. 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.
	Fig 5. Activated β-catenin is associated with JNK pathway activation.(A) Representative whole-mount immunofluorescence images and (B) quantification of 5-day-old control sibling and transgenic larvae treated for 2 days with 0.5% DMSO alone, 5 μM SP600125, or 5 μM EMD 420123 and stained with antibodies against phospho-c-Jun (left panels) and TO-PRO nuclear stain (right panels). Livers are outlined in white. Scale bar, 20 μm. N values are shown above the x-axis. Drug-treated zebrafish were compared to DMSO-treated siblings with the same genotype using 2-way ANOVA. **, p<0.001. (C) Representative photographs of zebrafish control and Tg(fabp10a:pt-β-cat) livers (left) and human HCC without (middle) and with (right) activated β-catenin, stained with anti-phospho-c-Jun antibodies. For zebrafish, 6 out of 8 (75%) Tg(fabp10a:pt-β-cat) livers with HCC contained one or more foci with moderate or high nuclear phospho-c-Jun staining (phospho-c-Jun positive), while all non-transgenic control livers (N = 7) without activated β-catenin showed absent or low staining (phospho-c-Jun negative). For human HCC, the number of cases exhibiting each staining pattern and the total number of cases with a given β-catenin activation status are shown at the bottom right of each picture. Images counterstained with hematoxylin. Scale bars, 20 μm. (D) Normalized jun mRNA expression in control sibling and transgenic adult zebrafish livers. Three technical replicates were performed for each sample. , p<0.05, Mann-Whitney test. N values are shown above the x-axis. (E) Normalized JUN mRNA expression for human HCC with low GLUL and high GLUL expression. **, p<0.001, Mann-Whitney test. (F) Graph showing dose of JNK inhibitor CC401 at which cell viability of human cancer cell lines was decreased by 50% (GI50), ± SEM. Number of replicates for each cell line is shown above the x-axis. , p<0.05, unpaired t-test.
	Fig 6. Antidepressants decrease β-catenin-induced liver enlargement and tumorigenesis.(A-B) Graphs showing mean liver size ± SEM of 6-day-old control sibling and transgenic zebrafish larvae treated for 3 days with amitriptyline (A) or paroxetine (B) 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<0.01; , p<0.001. (C) Graph showing mean liver size ± SEM of 6-day-old control sibling and transgenic zebrafish larvae treated for 3 days with 0.5% DMSO (-), 20 μM amitriptyline (AMI), 2 μM SP600125 (SP), or both drugs combined (AMI+SP). N values are shown above the x-axis. Asterisks indicate p-values for 2-way ANOVA comparing each group of transgenic zebrafish to AMI+SP group: , p<0.01; ***, p<0.001. N.S., no significant difference between groups of non-transgenic zebrafish (2-way ANOVA). (D) Representative images of control, non-hydrodynamically transfected (non-HDT) mouse livers (left) and mouse liver tumors induced by hydrodynamic transfection of activated β-catenin and Met (Met/β-cat HDT). Mice were treated with saccharine alone (vehicle only, top row) or amitriptyline plus saccharine (bottom row). Scale bars, 1 cm. (E) Graph showing mean liver-to-body mass ratios ± SEM for non-HDT and HDT-Met/β-cat mice treated with saccharine alone (vehicle) or amitriptyline plus saccharine (+Ami). P values calculated with Mann-Whitney test. N values are shown above the x-axis. (F) Representative hematoxylin-and-eosin-stained, Ki-67-labeled, and TUNEL-labeled images from vehicle- and amitriptyline-treated mice. Ki-67 and TUNEL stainings were performed using 3, 3'-diaminobenzidine (DAB) substrate, so positive-staining cells are brown, and hematoxylin counterstain to highlight nuclei and other basophilic structures in blue. (G-H) Graphs showing mean ± SEM of Ki-67-positive (G) or TUNEL-positive (H) cells per high-power field. P values calculated with Mann-Whitney test. N values are shown above the x-axis.