PAX3-FOXO1 transgenic zebrafish models identify HES3 as a mediator of rhabdomyosarcoma tumorigenesis

Alveolar rhabdomyosarcoma is a pediatric soft-tissue sarcoma caused by PAX3/7-FOXO1 fusion oncogenes and is characterized by impaired skeletal muscle development. We developed human PAX3-FOXO1 -driven zebrafish models of tumorigenesis and found that PAX3-FOXO1 exhibits discrete cell lineage susceptibility and transformation. Tumors developed by 1.6–19 months and were primitive neuroectodermal tumors or rhabdomyosarcoma. We applied this PAX3-FOXO1 transgenic zebrafish model to study how PAX3-FOXO1 leverages early developmental pathways for oncogenesis and found that her3 is a unique target. Ectopic expression of the her3 human ortholog, HES3, inhibits myogenesis in zebrafish and mammalian cells, recapitulating the arrested muscle development characteristic of rhabdomyosarcoma. In patients, HES3 is overexpressed in fusion-positive versus fusion-negative tumors. Finally, HES3 overexpression is associated with reduced survival in patients in the context of the fusion. Our novel zebrafish rhabdomyosarcoma model identifies a new PAX3-FOXO1 target, her3/HES3, that contributes to impaired myogenic differentiation and has prognostic significance in human disease.

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The LIM-only protein FHL2 interacts with β-catenin and promotes differentiation of mouse myoblasts

The Journal of Cell Biology ◽

10.1083/jcb.200202075 ◽

2002 ◽

Vol 159 (1) ◽

pp. 113-122 ◽

Cited By ~ 98

Author(s):

Bernd Martin ◽

Richard Schneider ◽

Stefanie Janetzky ◽

Zoe Waibler ◽

Petra Pandur ◽

...

Keyword(s):

Mammalian Cells ◽

Muscle Development ◽

Target Genes ◽

Myogenic Differentiation ◽

Myoblast Differentiation ◽

Lim Domains ◽

Vitro Binding ◽

Specific Proteins

FHL2 is a LIM-domain protein expressed in myoblasts but down-regulated in malignant rhabdomyosarcoma cells, suggesting an important role of FHL2 in muscle development. To investigate the importance of FHL2 during myoblast differentiation, we performed a yeast two-hybrid screen using a cDNA library derived from myoblasts induced for differentiation. We identified β-catenin as a novel interaction partner of FHL2 and confirmed the specificity of association by direct in vitro binding tests and coimmunoprecipitation assays from cell lysates. Deletion analysis of both proteins revealed that the NH2-terminal part of β-catenin is sufficient for binding in yeast, but addition of the first armadillo repeat is necessary for binding FHL2 in mammalian cells, whereas the presence of all four LIM domains of FHL2 is needed for the interaction. Expression of FHL2 counteracts β-catenin–mediated activation of a TCF/LEF-dependent reporter gene in a dose-dependent and muscle cell–specific manner. After injection into Xenopus embryos, FHL2 inhibited the β-catenin–induced axis duplication. C2C12 mouse myoblasts stably expressing FHL2 show increased myogenic differentiation reflected by accelerated myotube formation and expression of muscle-specific proteins. These data imply that FHL2 is a muscle-specific repressor of LEF/TCF target genes and promotes myogenic differentiation by interacting with β-catenin.

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HES6 acts as a transcriptional repressor in myoblasts and can induce the myogenic differentiation program

The Journal of Cell Biology ◽

10.1083/jcb.200104058 ◽

2001 ◽

Vol 154 (6) ◽

pp. 1161-1172 ◽

Cited By ~ 39

Author(s):

Xiangming Gao ◽

Tanya Chandra ◽

Michel-Olivier Gratton ◽

Isabelle Quélo ◽

Josée Prud'homme ◽

...

Keyword(s):

Transcriptional Repression ◽

Mammalian Cells ◽

Muscle Development ◽

Myogenic Differentiation ◽

Constitutive Expression ◽

Development Program ◽

Dominant Negative ◽

Functional Roles ◽

Helix Loop Helix ◽

Enhancer Of Split

HES6 is a novel member of the family of basic helix–loop–helix mammalian homologues of Drosophila Hairy and Enhancer of split. We have analyzed the biochemical and functional roles of HES6 in myoblasts. HES6 interacted with the corepressor transducin-like Enhancer of split 1 in yeast and mammalian cells through its WRPW COOH-terminal motif. HES6 repressed transcription from an N box–containing template and also when tethered to DNA through the GAL4 DNA binding domain. On N box–containing promoters, HES6 cooperated with HES1 to achieve maximal repression. An HES6–VP16 activation domain fusion protein activated the N box–containing reporter, confirming that HES6 bound the N box in muscle cells. The expression of HES6 was induced when myoblasts fused to become differentiated myotubes. Constitutive expression of HES6 in myoblasts inhibited expression of MyoR, a repressor of myogenesis, and induced differentiation, as evidenced by fusion into myotubes and expression of the muscle marker myosin heavy chain. Reciprocally, blocking endogenous HES6 function by using a WRPW-deleted dominant negative HES6 mutant led to increased expression of MyoR and completely blocked the muscle development program. Our results show that HES6 is an important regulator of myogenesis and suggest that MyoR is a target for HES6-dependent transcriptional repression.

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Coupling of DNA Synthesis and Histone Synthesis in S Phase Independent of Cyclin/cdk2 Activity

Molecular and Cellular Biology ◽

10.1128/mcb.22.21.7459-7472.2002 ◽

2002 ◽

Vol 22 (21) ◽

pp. 7459-7472 ◽

Cited By ~ 119

Author(s):

David M. Nelson ◽

Xiaofen Ye ◽

Caitlin Hall ◽

Hidelita Santos ◽

Tianlin Ma ◽

...

Keyword(s):

Dna Synthesis ◽

Mammalian Cells ◽

Ectopic Expression ◽

S Phase ◽

Phase Coupling ◽

Cdk2 Activity ◽

Human Ortholog ◽

Histone Synthesis ◽

Inhibition Of Dna Synthesis ◽

Histone Gene Transcription

ABSTRACT DNA and histone synthesis are both triggered at the beginning of S phase by cyclin/cdk2 activity. Previous studies showed that inhibition of DNA synthesis with hydroxyurea or cytosine arabinoside (AraC) triggers a concerted repression of histone synthesis, indicating that sustained histone synthesis depends on continued DNA synthesis. Here we show that ectopic expression of HIRA, the likely human ortholog of two cell cycle-regulated repressors of histone gene transcription in yeast (Hir1p and Hir2p), represses transcription of histones and that this, in turn, triggers a concerted block of DNA synthesis. Thus, in mammalian cells sustained DNA synthesis and histone synthesis are mutually dependent on each other during S phase. Although cyclin/cdk2 activity drives activation of both DNA and histone synthesis at the G1/S transition of cycling cells, concerted repression of DNA or histone synthesis in response to inhibition of either one of these is not accompanied by prolonged inhibition of cyclin A/cdk2 or E/cdk2 activity. Therefore, during S phase coupling of DNA and histone synthesis occurs, at least in part, through a mechanism that is independent of cyclin/cdk2 activity. Coupling of DNA and histone synthesis in S phase presumably contributes to the prompt and orderly assembly of newly replicated DNA into chromatin.

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Myogenic regulatory transcription factors regulate growth in rhabdomyosarcoma

eLife ◽

10.7554/elife.19214 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 25

Author(s):

Inês M Tenente ◽

Madeline N Hayes ◽

Myron S Ignatius ◽

Karin McCarthy ◽

Marielle Yohe ◽

...

Keyword(s):

Transcription Factors ◽

Tumor Growth ◽

Cell Cycle Progression ◽

Muscle Development ◽

Regulatory Elements ◽

Transgenic Zebrafish ◽

Mechanistic Studies ◽

Zebrafish Model ◽

Cycle Progression ◽

Dna Regulatory Elements

Rhabdomyosarcoma (RMS) is a pediatric malignacy of muscle with myogenic regulatory transcription factors MYOD and MYF5 being expressed in this disease. Consensus in the field has been that expression of these factors likely reflects the target cell of transformation rather than being required for continued tumor growth. Here, we used a transgenic zebrafish model to show that Myf5 is sufficient to confer tumor-propagating potential to RMS cells and caused tumors to initiate earlier and have higher penetrance. Analysis of human RMS revealed that MYF5 and MYOD are mutually-exclusively expressed and each is required for sustained tumor growth. ChIP-seq and mechanistic studies in human RMS uncovered that MYF5 and MYOD bind common DNA regulatory elements to alter transcription of genes that regulate muscle development and cell cycle progression. Our data support unappreciated and dominant oncogenic roles for MYF5 and MYOD convergence on common transcriptional targets to regulate human RMS growth.

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MicroRNA-21 Plays Multiple Oncometabolic Roles in the Process of NAFLD-Related Hepatocellular Carcinoma via PI3K/AKT, TGF-β, and STAT3 Signaling

Cancers ◽

10.3390/cancers13050940 ◽

2021 ◽

Vol 13 (5) ◽

pp. 940

Author(s):

Chi-Yu Lai ◽

Kun-Yun Yeh ◽

Chiu-Ya Lin ◽

Yang-Wen Hsieh ◽

Hsin-Hung Lai ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Signaling Pathways ◽

Stellate Cell ◽

Human Cancer ◽

Tumor Formation ◽

Transgenic Zebrafish ◽

Zebrafish Model ◽

Lipid Metabolism Disorder ◽

Full Spectrum ◽

Stat3 Signaling

MicroRNA-21 (miR-21) is one of the most frequently upregulated miRNAs in liver diseases such as nonalcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC). However, mechanistic pathways that connect NAFLD and HCC remain elusive. We developed a doxycycline (Dox)-inducible transgenic zebrafish model (LmiR21) which exhibited an upregulation of miR-21 in the liver, which in turn induced the full spectrum of NAFLD, including steatosis, inflammation, fibrosis, and HCC, in the LmiR21 fish. Diethylnitrosamine (DEN) treatment led to accelerated liver tumor formation and exacerbated their aggressiveness. Moreover, prolonged miR-21 expression for up to ten months induced nonalcoholic steatohepatitis (NASH)-related HCC (NAHCC). Immunoblotting and immunostaining confirmed the presence of miR-21 regulatory proteins (i.e., PTEN, SMAD7, p-AKT, p-SMAD3, and p-STAT3) in human nonviral HCC tissues and LmiR21 models. Thus, we demonstrated that miR-21 can induce NAHCC via at least three mechanisms: First, the occurrence of hepatic steatosis increases with the decrease of ptenb, pparaa, and activation of the PI3K/AKT pathway; second, miR-21 induces hepatic inflammation (or NASH) through an increase in inflammatory gene expression via STAT3 signaling pathways, and induces liver fibrosis through hepatic stellate cell (HSC) activation and collagen deposition via TGF-β/Smad3/Smad7 signaling pathways; finally, oncogenic activation of Smad3/Stat3 signaling pathways induces HCC. Our LmiR21 models showed similar molecular pathology to the human cancer samples in terms of initiation of lipid metabolism disorder, inflammation, fibrosis and activation of the PI3K/AKT, TGF-β/SMADs and STAT3 (PTS) oncogenic signaling pathways. Our findings indicate that miR-21 plays critical roles in the mechanistic perspectives of NAHCC development via the PTS signaling networks.

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Author Correction: Stimulation of hepatocarcinogenesis by activated cholangiocytes via Il17a/f1 pathway in kras transgenic zebrafish model

Scientific Reports ◽

10.1038/s41598-021-92144-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Mohamed Helal ◽

Chuan Yan ◽

Zhiyuan Gong

Keyword(s):

Transgenic Zebrafish ◽

Zebrafish Model ◽

Stimulation Of

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Modulation of Innate Immune Toxicity by Silver Nanoparticle Exposure and the Preventive Effects of Pterostilbene

International Journal of Molecular Sciences ◽

10.3390/ijms22052536 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2536

Author(s):

Rong-Jane Chen ◽

Chiao-Ching Huang ◽

Rosita Pranata ◽

Yu-Hsuan Lee ◽

Yu-Ying Chen ◽

...

Keyword(s):

Silver Nanoparticles ◽

Good Alternative ◽

Innate Immune ◽

Toxic Effects ◽

Transgenic Zebrafish ◽

Zebrafish Model ◽

Cytokines And Chemokines ◽

Living Organisms ◽

And Function ◽

Immune Toxicity

Silver nanoparticles pose a potential risk to ecosystems and living organisms due to their widespread use in various fields and subsequent gradual release into the environment. Only a few studies have investigated the effects of silver nanoparticles (AgNPs) toxicity on immunological functions. Furthermore, these toxic effects have not been fully explored. Recent studies have indicated that zebrafish are considered a good alternative model for testing toxicity and for evaluating immunological toxicity. Therefore, the purpose of this study was to investigate the toxicity effects of AgNPs on innate immunity using a zebrafish model and to investigate whether the natural compound pterostilbene (PTE) could provide protection against AgNPs-induced immunotoxicity. Wild type and neutrophil- and macrophage-transgenic zebrafish lines were used in the experiments. The results indicated that the exposure to AgNPs induced toxic effects including death, malformation and the innate immune toxicity of zebrafish. In addition, AgNPs affect the number and function of neutrophils and macrophages. The expression of immune-related cytokines and chemokines was also affected. Notably, the addition of PTE could activate immune cells and promote their accumulation in injured areas in zebrafish, thereby reducing the damage caused by AgNPs. In conclusion, AgNPs may induce innate immune toxicity and PTE could ameliorate this toxicity.

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Ectopic Expression of Inhibitors of Protein Phosphatase Type 1 (PP1) Can Be Used to Analyze Roles of PP1 in Drosophila Development

Genetics ◽

10.1093/genetics/164.1.235 ◽

2003 ◽

Vol 164 (1) ◽

pp. 235-245

Author(s):

Daimark Bennett ◽

Balázs Szöőr ◽

Sascha Gross ◽

Natalia Vereshchagina ◽

Luke Alphey

Keyword(s):

Protein Phosphatase ◽

Muscle Development ◽

Ectopic Expression ◽

High Specificity ◽

Type I ◽

Protein Phosphatase Type 1 ◽

Mitotic Figures ◽

Protein Phosphatase Type

Abstract We have identified two proteins that bind with high specificity to type 1 serine/threonine protein phosphatase (PP1) and have exploited their inhibitory properties to develop an efficient and flexible strategy for conditional inactivation of PP1 in vivo. We show that modest overexpression of Drosophila homologs of I-2 and NIPP1 (I-2Dm and NIPP1Dm) reduces the level of PP1 activity and phenotypically resembles known PP1 mutants. These phenotypes, which include lethality, abnormal mitotic figures, and defects in muscle development, are suppressed by coexpression of PP1, indicating that the effect is due specifically to loss of PP1 activity. Reactivation of I-2Dm:PP1c complexes suggests that inhibition of PP1 activity in vivo does not result in a compensating increase in synthesis of active PP1. PP1 mutants enhance the wing overgrowth phenotype caused by ectopic expression of the type II TGFβ superfamily signaling receptor Punt. Using I-2Dm, which has a less severe effect than NIPP1Dm, we show that lowering the level of PP1 activity specifically in cells overexpressing Punt is sufficient for wing overgrowth and that the interaction between PP1 and Punt requires the type I receptor Thick-veins (Tkv) but is not strongly sensitive to the level of the ligand, Decapentaplegic (Dpp), nor to that of the other type I receptors. This is consistent with a role for PP1 in antagonizing Punt by preventing phosphorylation of Tkv. These studies demonstrate that inhibitors of PP1 can be used in a tissue- and developmental-specific manner to examine the developmental roles of PP1.

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Development of BCR-ABL1 Transgenic Zebrafish Model Reproducing Chronic Myeloid Leukemia (CML) Like-Disease and Providing a New Insight into CML Mechanisms

Cells ◽

10.3390/cells10020445 ◽

2021 ◽

Vol 10 (2) ◽

pp. 445

Author(s):

Daniela Zizioli ◽

Simona Bernardi ◽

Marco Varinelli ◽

Mirko Farina ◽

Luca Mignani ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Fusion Gene ◽

Philadelphia Chromosome ◽

Leukemic Cells ◽

Transgenic Zebrafish ◽

Hematopoietic Tissue ◽

Zebrafish Model ◽

Altered Expression

Zebrafish has proven to be a versatile and reliable experimental in vivo tool to study human hematopoiesis and model hematological malignancies. Transgenic technologies enable the generation of specific leukemia types by the expression of human oncogenes under specific promoters. Using this technology, a variety of myeloid and lymphoid malignancies zebrafish models have been described. Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasia characterized by the BCR-ABL1 fusion gene, derived from the t (9;22) translocation causing the Philadelphia Chromosome (Ph). The BCR-ABL1 protein is a constitutively activated tyrosine kinas inducing the leukemogenesis and resulting in an accumulation of immature leukemic cells into bone marrow and peripheral blood. To model Ph+ CML, a transgenic zebrafish line expressing the human BCR-ABL1 was generated by the Gal4/UAS system, and then crossed with the hsp70-Gal4 transgenic line. The new line named (BCR-ABL1pUAS:CFP/hsp70-Gal4), presented altered expression of hematopoietic markers during embryonic development compared to controls and transgenic larvae showed proliferating hematopoietic cells in the caudal hematopoietic tissue (CHT). The present transgenic zebrafish would be a robust CML model and a high-throughput drug screening tool.

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