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1.
Mol Cell Biol ; 43(11): 547-565, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37882064

RESUMO

Rhabdomyosarcoma (RMS) is a pediatric malignancy of the muscle with characteristics of cells blocked in differentiation. NOTCH1 is an oncogene that promotes self-renewal and blocks differentiation in the fusion negative-RMS sub-type. However, how NOTCH1 expression is transcriptionally maintained in tumors is unknown. Analyses of SNAI2 and CTCF chromatin binding and HiC analyses revealed a conserved SNAI2/CTCF overlapping peak downstream of the NOTCH1 locus marking a sub-topologically associating domain (TAD) boundary. Deletion of the SNAI2-CTCF peak showed that it is essential for NOTCH1 expression and viability of FN-RMS cells. Reintroducing constitutively activated NOTCH1-ΔE in cells with the SNAI2-CTCF peak deleted restored cell-viability. Ablation of SNAI2 using CRISPR/Cas9 reagents resulted in the loss of majority of RD and SMS-CTR FN-RMS cells. However, the few surviving clones that repopulate cultures have recovered NOTCH1. Cells that re-establish NOTCH1 expression after SNAI2 ablation are unable to differentiate robustly as SNAI2 shRNA knockdown cells; yet, SNAI2-ablated cells continued to be exquisitely sensitive to ionizing radiation. Thus, we have uncovered a novel mechanism by which SNAI2 and CTCF maintenance of a sub-TAD boundary promotes rather than represses NOTCH1 expression. Further, we demonstrate that SNAI2 suppression of apoptosis post-radiation is independent of SNAI2/NOTCH1 effects on self-renewal and differentiation.


Assuntos
Cromatina , Rabdomiossarcoma , Criança , Humanos , Fator de Ligação a CCCTC/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Rabdomiossarcoma/genética , RNA Interferente Pequeno/genética , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo
2.
Elife ; 122023 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-37266578

RESUMO

In embryonal rhabdomyosarcoma (ERMS) and generally in sarcomas, the role of wild-type and loss- or gain-of-function TP53 mutations remains largely undefined. Eliminating mutant or restoring wild-type p53 is challenging; nevertheless, understanding p53 variant effects on tumorigenesis remains central to realizing better treatment outcomes. In ERMS, >70% of patients retain wild-type TP53, yet mutations when present are associated with worse prognosis. Employing a kRASG12D-driven ERMS tumor model and tp53 null (tp53-/-) zebrafish, we define wild-type and patient-specific TP53 mutant effects on tumorigenesis. We demonstrate that tp53 is a major suppressor of tumorigenesis, where tp53 loss expands tumor initiation from <35% to >97% of animals. Characterizing three patient-specific alleles reveals that TP53C176F partially retains wild-type p53 apoptotic activity that can be exploited, whereas TP53P153Δ and TP53Y220C encode two structurally related proteins with gain-of-function effects that predispose to head musculature ERMS. TP53P153Δ unexpectedly also predisposes to hedgehog-expressing medulloblastomas in the kRASG12D-driven ERMS-model.


Assuntos
Neoplasias Cerebelares , Rabdomiossarcoma Embrionário , Animais , Carcinogênese , Mutação , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Rabdomiossarcoma Embrionário/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
3.
Mol Cancer Ther ; 22(1): 123-134, 2023 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-36162055

RESUMO

In fusion-negative rhabdomyosarcoma (FN-RMS), a pediatric malignancy with skeletal muscle characteristics, >90% of high-risk patients have mutations that activate the RAS/MEK signaling pathway. We recently discovered that SNAI2, in addition to blocking myogenic differentiation downstream of MEK signaling in FN-RMS, represses proapoptotic BIM expression to protect RMS tumors from ionizing radiation (IR). As clinically relevant concentrations of the MEK inhibitor trametinib elicit poor responses in preclinical xenograft models, we investigated the utility of low-dose trametinib in combination with IR for the treatment of RAS-mutant FN-RMS. We hypothesized that trametinib would sensitize FN-RMS to IR through its downregulation of SNAI2 expression. While we observed little to no difference in myogenic differentiation or cell survival with trametinib treatment alone, robust differentiation and reduced survival were observed after IR. In addition, IR-induced apoptosis was significantly increased in FN-RMS cells treated concurrently with trametinib, as was increased BIM expression. SNAI2's role in these processes was established using overexpression rescue experiments, where overexpression of SNAI2 prevented IR-induced myogenic differentiation and apoptosis. Moreover, combining MEK inhibitor with IR resulted in complete tumor regression and a 2- to 4-week delay in event-free survival (EFS) in preclinical xenograft and patient-derived xenograft models. Our findings demonstrate that the combination of MEK inhibition and IR results in robust differentiation and apoptosis, due to the reduction of SNAI2, which leads to extended EFS in FN-RMS. SNAI2 thus is a potential biomarker of IR insensitivity and target for future therapies to sensitize aggressive sarcomas to IR.


Assuntos
Rabdomiossarcoma , Criança , Humanos , Rabdomiossarcoma/tratamento farmacológico , Rabdomiossarcoma/genética , Rabdomiossarcoma/radioterapia , Diferenciação Celular , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Quinases de Proteína Quinase Ativadas por Mitógeno , Linhagem Celular Tumoral , Fatores de Transcrição da Família Snail
4.
Mol Cancer Ther ; 21(1): 170-183, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34737198

RESUMO

Oncogenic RAS signaling is an attractive target for fusion-negative rhabdomyosarcoma (FN-RMS). Our study validates the role of the ERK MAPK effector pathway in mediating RAS dependency in a panel of H/NRASQ61X mutant RMS cells and correlates in vivo efficacy of the MEK inhibitor trametinib with pharmacodynamics of ERK activity. A screen is used to identify trametinib-sensitizing targets, and combinations are evaluated in cells and tumor xenografts. We find that the ERK MAPK pathway is central to H/NRASQ61X dependency in RMS cells; however, there is poor in vivo response to clinically relevant exposures with trametinib, which correlates with inefficient suppression of ERK activity. CRISPR screening points to vertical inhibition of the RAF-MEK-ERK cascade by cosuppression of MEK and either CRAF or ERK. CRAF is central to rebound pathway activation following MEK or ERK inhibition. Concurrent CRAF suppression and MEK or ERK inhibition, or concurrent pan-RAF and MEK/ERK inhibition (pan-RAFi + MEKi/ERKi), or concurrent MEK and ERK inhibition (MEKi + ERKi) all synergistically block ERK activity and induce myogenic differentiation and apoptosis. In vivo assessment of pan-RAFi + ERKi or MEKi + ERKi potently suppress growth of H/NRASQ61X RMS tumor xenografts, with pan-RAFi + ERKi being more effective and better tolerated. We conclude that CRAF reactivation limits the activity of single-agent MEK/ERK inhibitors in FN-RMS. Vertical targeting of the RAF-MEK-ERK cascade and particularly cotargeting of CRAF and MEK or ERK, or the combination of pan-RAF inhibitors with MEK or ERK inhibitors, have synergistic activity and potently suppress H/NRASQ61X mutant RMS tumor growth.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Rabdomiossarcoma/genética , Animais , Apoptose , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Genes ras , Humanos , Camundongos , Rabdomiossarcoma/patologia , Transfecção
5.
Cancer Res ; 81(21): 5451-5463, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34462275

RESUMO

Ionizing radiation (IR) and chemotherapy are mainstays of treatment for patients with rhabdomyosarcoma, yet the molecular mechanisms that underlie the success or failure of radiotherapy remain unclear. The transcriptional repressor SNAI2 was previously identified as a key regulator of IR sensitivity in normal and malignant stem cells through its repression of the proapoptotic BH3-only gene PUMA/BBC3. Here, we demonstrate a clear correlation between SNAI2 expression levels and radiosensitivity across multiple rhabdomyosarcoma cell lines. Modulating SNAI2 levels in rhabdomyosarcoma cells through its overexpression or knockdown altered radiosensitivity in vitro and in vivo. SNAI2 expression reliably promoted overall cell growth and inhibited mitochondrial apoptosis following exposure to IR, with either variable or minimal effects on differentiation and senescence, respectively. Importantly, SNAI2 knockdown increased expression of the proapoptotic BH3-only gene BIM, and chromatin immunoprecipitation sequencing experiments established that SNAI2 is a direct repressor of BIM/BCL2L11. Because the p53 pathway is nonfunctional in the rhabdomyosarcoma cells used in this study, we have identified a new, p53-independent SNAI2/BIM signaling axis that could potentially predict clinical responses to IR treatment and be exploited to improve rhabdomyosarcoma therapy. SIGNIFICANCE: SNAI2 is identified as a major regulator of radiation-induced apoptosis in rhabdomyosarcoma through previously unknown mechanisms independent of p53.


Assuntos
Proteína 11 Semelhante a Bcl-2/antagonistas & inibidores , Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Radiação Ionizante , Rabdomiossarcoma/prevenção & controle , Fatores de Transcrição da Família Snail/metabolismo , Animais , Apoptose , Proteína 11 Semelhante a Bcl-2/genética , Proteína 11 Semelhante a Bcl-2/metabolismo , Biomarcadores Tumorais/genética , Ciclo Celular , Movimento Celular , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos SCID , RNA-Seq , Rabdomiossarcoma/etiologia , Rabdomiossarcoma/patologia , Fatores de Transcrição da Família Snail/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Zebrafish ; 18(4): 293-296, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34030492

RESUMO

Angiosarcoma is a clinically aggressive tumor with a high rate of mortality. It can arise in vascular or lymphatic tissues, involve any part of the body, and aggressively spread locally or metastasize. Angiosarcomas spontaneously develop in the tp53 deleted (tp53del/del) zebrafish mutant. However, established protocols for tumor dissection and transplantation of single cell suspensions of angiosarcoma tumors result in inferior implantation rates. To resolve these complications, we developed a new tumor grafting technique for engraftment of angiosarcoma and similar tumors in zebrafish, which maintains the tumor microenvironment and has superior rates of engraftment.


Assuntos
Hemangiossarcoma , Transplante de Neoplasias , Peixe-Zebra , Animais , Modelos Animais de Doenças , Hemangiossarcoma/patologia , Suspensões , Microambiente Tumoral
7.
Nat Commun ; 12(1): 192, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33420019

RESUMO

Rhabdomyosarcoma (RMS) is an aggressive pediatric malignancy of the muscle, that includes Fusion Positive (FP)-RMS harboring PAX3/7-FOXO1 and Fusion Negative (FN)-RMS commonly with RAS pathway mutations. RMS express myogenic master transcription factors MYOD and MYOG yet are unable to terminally differentiate. Here, we report that SNAI2 is highly expressed in FN-RMS, is oncogenic, blocks myogenic differentiation, and promotes growth. MYOD activates SNAI2 transcription via super enhancers with striped 3D contact architecture. Genome wide chromatin binding analysis demonstrates that SNAI2 preferentially binds enhancer elements and competes with MYOD at a subset of myogenic enhancers required for terminal differentiation. SNAI2 also suppresses expression of a muscle differentiation program modulated by MYOG, MEF2, and CDKN1A. Further, RAS/MEK-signaling modulates SNAI2 levels and binding to chromatin, suggesting that the differentiation blockade by oncogenic RAS is mediated in part by SNAI2. Thus, an interplay between SNAI2, MYOD, and RAS prevents myogenic differentiation and promotes tumorigenesis.


Assuntos
Carcinogênese/metabolismo , Diferenciação Celular , Proteína MyoD/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Rabdomiossarcoma/genética , Rabdomiossarcoma/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Animais , Carcinogênese/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Fatores de Transcrição MEF2/metabolismo , Masculino , Camundongos , Camundongos SCID , Desenvolvimento Muscular/genética , Proteína MyoD/genética , Miogenina/metabolismo , Proteínas de Fusão Oncogênica/genética , Oncogenes , Rabdomiossarcoma/patologia , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Embrionário/genética , Fatores de Transcrição da Família Snail/genética , Transcriptoma
8.
Pediatr Blood Cancer ; 66(10): e27869, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31222885

RESUMO

Overall survival rates for pediatric patients with high-risk or relapsed rhabdomyosarcoma (RMS) have not improved significantly since the 1980s. Recent studies have identified a number of targetable vulnerabilities in RMS, but these discoveries have infrequently translated into clinical trials. We propose streamlining the process by which agents are selected for clinical evaluation in RMS. We believe that strong consideration should be given to the development of combination therapies that add biologically targeted agents to conventional cytotoxic drugs. One example of this type of combination is the addition of the WEE1 inhibitor AZD1775 to the conventional cytotoxic chemotherapeutics, vincristine and irinotecan.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Desenvolvimento de Medicamentos/métodos , Descoberta de Drogas/métodos , Rabdomiossarcoma , Criança , Humanos , Projetos de Pesquisa
9.
Elife ; 72018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30192230

RESUMO

The TP53 tumor-suppressor gene is mutated in >50% of human tumors and Li-Fraumeni patients with germ line inactivation are predisposed to developing cancer. Here, we generated tp53 deleted zebrafish that spontaneously develop malignant peripheral nerve-sheath tumors, angiosarcomas, germ cell tumors, and an aggressive Natural Killer cell-like leukemia for which no animal model has been developed. Because the tp53 deletion was generated in syngeneic zebrafish, engraftment of fluorescent-labeled tumors could be dynamically visualized over time. Importantly, engrafted tumors shared gene expression signatures with predicted cells of origin in human tissue. Finally, we showed that tp53del/del enhanced invasion and metastasis in kRASG12D-induced embryonal rhabdomyosarcoma (ERMS), but did not alter the overall frequency of cancer stem cells, suggesting novel pro-metastatic roles for TP53 loss-of-function in human muscle tumors. In summary, we have developed a Li-Fraumeni zebrafish model that is amenable to large-scale transplantation and direct visualization of tumor growth in live animals.


Assuntos
Rabdomiossarcoma Embrionário/metabolismo , Rabdomiossarcoma Embrionário/patologia , Proteína Supressora de Tumor p53/deficiência , Peixe-Zebra/metabolismo , Animais , Contagem de Células , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Hemangiossarcoma/metabolismo , Hemangiossarcoma/patologia , Homozigoto , Leucemia/metabolismo , Leucemia/patologia , Invasividade Neoplásica , Metástase Neoplásica , Transplante de Neoplasias , Neoplasias Embrionárias de Células Germinativas/metabolismo , Neoplasias Embrionárias de Células Germinativas/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Rabdomiossarcoma Embrionário/genética , Análise de Sobrevida , Proteína Supressora de Tumor p53/metabolismo , Peixe-Zebra/genética
10.
Cell Stem Cell ; 22(3): 414-427.e6, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29499154

RESUMO

Tumor growth and relapse are driven by tumor propagating cells (TPCs). However, mechanisms regulating TPC fate choices, maintenance, and self-renewal are not fully understood. Here, we show that Van Gogh-like 2 (Vangl2), a core regulator of the non-canonical Wnt/planar cell polarity (Wnt/PCP) pathway, affects TPC self-renewal in rhabdomyosarcoma (RMS)-a pediatric cancer of muscle. VANGL2 is expressed in a majority of human RMS and within early mononuclear progenitor cells. VANGL2 depletion inhibited cell proliferation, reduced TPC numbers, and induced differentiation of human RMS in vitro and in mouse xenografts. Using a zebrafish model of embryonal rhabdomyosarcoma (ERMS), we determined that Vangl2 expression enriches for TPCs and promotes their self-renewal. Expression of constitutively active and dominant-negative isoforms of RHOA revealed that it acts downstream of VANGL2 to regulate proliferation and maintenance of TPCs in human RMS. Our studies offer insights into pathways that control TPCs and identify new potential therapeutic targets.


Assuntos
Autorrenovação Celular , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Células-Tronco Neoplásicas/patologia , Rabdomiossarcoma/patologia , Transdução de Sinais , Proteínas de Peixe-Zebra/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Células-Tronco Neoplásicas/metabolismo , Rabdomiossarcoma/genética , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Cell Rep ; 19(11): 2304-2318, 2017 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-28614716

RESUMO

Tumor-propagating cells (TPCs) share self-renewal properties with normal stem cells and drive continued tumor growth. However, mechanisms regulating TPC self-renewal are largely unknown, especially in embryonal rhabdomyosarcoma (ERMS)-a common pediatric cancer of muscle. Here, we used a zebrafish transgenic model of ERMS to identify a role for intracellular NOTCH1 (ICN1) in increasing TPCs by 23-fold. ICN1 expanded TPCs by enabling the de-differentiation of zebrafish ERMS cells into self-renewing myf5+ TPCs, breaking the rigid differentiation hierarchies reported in normal muscle. ICN1 also had conserved roles in regulating human ERMS self-renewal and growth. Mechanistically, ICN1 upregulated expression of SNAIL1, a transcriptional repressor, to increase TPC number in human ERMS and to block muscle differentiation through suppressing MEF2C, a myogenic differentiation transcription factor. Our data implicate the NOTCH1/SNAI1/MEF2C signaling axis as a major determinant of TPC self-renewal and differentiation in ERMS, raising hope of therapeutically targeting this pathway in the future.


Assuntos
Fatores de Transcrição MEF2/metabolismo , Receptor Notch1/metabolismo , Rabdomiossarcoma Embrionário/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Animais , Diferenciação Celular/fisiologia , Humanos , Rabdomiossarcoma Embrionário/patologia , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/metabolismo , Peixe-Zebra
12.
Elife ; 62017 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-28080960

RESUMO

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.


Assuntos
Proteína MyoD/metabolismo , Fator Regulador Miogênico 5/metabolismo , Rabdomiossarcoma/fisiopatologia , Transcrição Gênica , Animais , Animais Geneticamente Modificados , Imunoprecipitação da Cromatina , Humanos , Análise de Sequência de DNA , Peixe-Zebra
13.
Adv Exp Med Biol ; 916: 219-37, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27165356

RESUMO

Zebrafish cancer models have greatly advanced our understanding of malignancy in humans. This is made possible due to the unique advantages of the zebrafish model including ex vivo development and large clutch sizes, which enable large-scale genetic and chemical screens. Transparency of the embryo and the creation of adult zebrafish devoid of pigmentation (casper) have permitted unprecedented ability to dynamically visualize cancer progression in live animals. When coupled with fluorescent reporters and transgenic approaches that drive oncogenesis, it is now possible to label entire or subpopulations of cancer cells and follow cancer growth in near real-time. Here, we will highlight aspects of in vivo imaging using the zebrafish and how it has enhanced our understanding of the fundamental aspects of tumor initiation, self-renewal, neovascularization, tumor cell heterogeneity, invasion and metastasis. Importantly, we will highlight the contribution of cancer imaging in zebrafish for drug discovery.


Assuntos
Diagnóstico por Imagem , Modelos Animais de Doenças , Neoplasias/patologia , Animais , Animais Geneticamente Modificados , Progressão da Doença , Neoplasias/irrigação sanguínea , Células-Tronco Neoplásicas/patologia , Neovascularização Patológica , Peixe-Zebra/embriologia
14.
Nat Commun ; 7: 10358, 2016 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-26790525

RESUMO

Cancers contain a wide diversity of cell types that are defined by differentiation states, genetic mutations and altered epigenetic programmes that impart functional diversity to individual cells. Elevated tumour cell heterogeneity is linked with progression, therapy resistance and relapse. Yet, imaging of tumour cell heterogeneity and the hallmarks of cancer has been a technical and biological challenge. Here we develop optically clear immune-compromised rag2(E450fs) (casper) zebrafish for optimized cell transplantation and direct visualization of fluorescently labelled cancer cells at single-cell resolution. Tumour engraftment permits dynamic imaging of neovascularization, niche partitioning of tumour-propagating cells in embryonal rhabdomyosarcoma, emergence of clonal dominance in T-cell acute lymphoblastic leukaemia and tumour evolution resulting in elevated growth and metastasis in BRAF(V600E)-driven melanoma. Cell transplantation approaches using optically clear immune-compromised zebrafish provide unique opportunities to uncover biology underlying cancer and to dynamically visualize cancer processes at single-cell resolution in vivo.


Assuntos
Rastreamento de Células/métodos , Melanoma/química , Melanoma/imunologia , Animais , Rastreamento de Células/instrumentação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Modelos Animais de Doenças , Progressão da Doença , Humanos , Hospedeiro Imunocomprometido , Melanoma/genética , Melanoma/patologia , Metástase Neoplásica , Transplante de Neoplasias , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/imunologia
15.
Nat Methods ; 11(8): 821-4, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25042784

RESUMO

Cell transplantation into adult zebrafish has lagged behind mouse models owing to the lack of immunocompromised strains. Here we have created rag2(E450fs) mutant zebrafish that have reduced numbers of functional T and B cells but are viable and fecund. Mutant fish engraft muscle, blood stem cells and various cancers. rag2(E450fs) mutant zebrafish are the first immunocompromised zebrafish model that permits robust, long-term engraftment of multiple tissues and cancer.


Assuntos
Transplante de Células , Proteínas de Ligação a DNA/genética , Mutação , Peixe-Zebra/genética , Idoso , Animais , Humanos
16.
Proc Natl Acad Sci U S A ; 111(14): 5349-54, 2014 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-24706870

RESUMO

Embryonal rhabdomyosarcoma (ERMS) is a common pediatric malignancy of muscle, with relapse being the major clinical challenge. Self-renewing tumor-propagating cells (TPCs) drive cancer relapse and are confined to a molecularly definable subset of ERMS cells. To identify drugs that suppress ERMS self-renewal and induce differentiation of TPCs, a large-scale chemical screen was completed. Glycogen synthase kinase 3 (GSK3) inhibitors were identified as potent suppressors of ERMS growth through inhibiting proliferation and inducing terminal differentiation of TPCs into myosin-expressing cells. In support of GSK3 inhibitors functioning through activation of the canonical WNT/ß-catenin pathway, recombinant WNT3A and stabilized ß-catenin also enhanced terminal differentiation of human ERMS cells. Treatment of ERMS-bearing zebrafish with GSK3 inhibitors activated the WNT/ß-catenin pathway, resulting in suppressed ERMS growth, depleted TPCs, and diminished self-renewal capacity in vivo. Activation of the canonical WNT/ß-catenin pathway also significantly reduced self-renewal of human ERMS, indicating a conserved function for this pathway in modulating ERMS self-renewal. In total, we have identified an unconventional tumor suppressive role for the canonical WNT/ß-catenin pathway in regulating self-renewal of ERMS and revealed therapeutic strategies to target differentiation of TPCs in ERMS.


Assuntos
Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Rabdomiossarcoma Embrionário/patologia , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo , Animais , Linhagem Celular , Humanos , Rabdomiossarcoma Embrionário/enzimologia , Rabdomiossarcoma Embrionário/metabolismo , Peixe-Zebra
17.
PLoS Genet ; 9(8): e1003727, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24009521

RESUMO

Human cancer genomes are highly complex, making it challenging to identify specific drivers of cancer growth, progression, and tumor maintenance. To bypass this obstacle, we have applied array comparative genomic hybridization (array CGH) to zebrafish embryonal rhabdomyosaroma (ERMS) and utilized cross-species comparison to rapidly identify genomic copy number aberrations and novel candidate oncogenes in human disease. Zebrafish ERMS contain small, focal regions of low-copy amplification. These same regions were commonly amplified in human disease. For example, 16 of 19 chromosomal gains identified in zebrafish ERMS also exhibited focal, low-copy gains in human disease. Genes found in amplified genomic regions were assessed for functional roles in promoting continued tumor growth in human and zebrafish ERMS--identifying critical genes associated with tumor maintenance. Knockdown studies identified important roles for Cyclin D2 (CCND2), Homeobox Protein C6 (HOXC6) and PlexinA1 (PLXNA1) in human ERMS cell proliferation. PLXNA1 knockdown also enhanced differentiation, reduced migration, and altered anchorage-independent growth. By contrast, chemical inhibition of vascular endothelial growth factor (VEGF) signaling reduced angiogenesis and tumor size in ERMS-bearing zebrafish. Importantly, VEGFA expression correlated with poor clinical outcome in patients with ERMS, implicating inhibitors of the VEGF pathway as a promising therapy for improving patient survival. Our results demonstrate the utility of array CGH and cross-species comparisons to identify candidate oncogenes essential for the pathogenesis of human cancer.


Assuntos
Hibridização Genômica Comparativa , Neoplasias/genética , Oncogenes , Rabdomiossarcoma Embrionário/genética , Peixe-Zebra/genética , Animais , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Humanos , Hibridização in Situ Fluorescente , Neoplasias/etiologia , Análise de Sequência com Séries de Oligonucleotídeos , Rabdomiossarcoma Embrionário/patologia
18.
PLoS One ; 8(5): e63218, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23667588

RESUMO

The regulation of gene expression is accomplished by both genetic and epigenetic means and is required for the precise control of the development of the neural crest. In hdac1(b382) mutants, craniofacial cartilage development is defective in two distinct ways. First, fewer hoxb3a, dlx2 and dlx3-expressing posterior branchial arch precursors are specified and many of those that are consequently undergo apoptosis. Second, in contrast, normal numbers of progenitors are present in the anterior mandibular and hyoid arches, but chondrocyte precursors fail to terminally differentiate. In the peripheral nervous system, there is a disruption of enteric, DRG and sympathetic neuron differentiation in hdac1(b382) mutants compared to wildtype embryos. Specifically, enteric and DRG-precursors differentiate into neurons in the anterior gut and trunk respectively, while enteric and DRG neurons are rarely present in the posterior gut and tail. Sympathetic neuron precursors are specified in hdac1(b382) mutants and they undergo generic neuronal differentiation but fail to undergo noradrenergic differentiation. Using the HDAC inhibitor TSA, we isolated enzyme activity and temporal requirements for HDAC function that reproduce hdac1(b382) defects in craniofacial and sympathetic neuron development. Our study reveals distinct functional and temporal requirements for zebrafish hdac1 during neural crest-derived craniofacial and peripheral neuron development.


Assuntos
Face/embriologia , Histona Desacetilase 1/metabolismo , Crista Neural/patologia , Neurônios/metabolismo , Crânio/embriologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Região Branquial/anormalidades , Região Branquial/embriologia , Região Branquial/patologia , Diferenciação Celular/efeitos dos fármacos , Anormalidades Craniofaciais/embriologia , Anormalidades Craniofaciais/patologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Face/anormalidades , Face/patologia , Histona Desacetilase 1/genética , Ácidos Hidroxâmicos/farmacologia , Osso Hioide/anormalidades , Osso Hioide/efeitos dos fármacos , Osso Hioide/embriologia , Osso Hioide/patologia , Mandíbula/anormalidades , Mandíbula/efeitos dos fármacos , Mandíbula/embriologia , Mandíbula/patologia , Mutação/genética , Crista Neural/efeitos dos fármacos , Crista Neural/embriologia , Crista Neural/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Sistema Nervoso Periférico/efeitos dos fármacos , Sistema Nervoso Periférico/embriologia , Sistema Nervoso Periférico/patologia , Fenótipo , Crânio/anormalidades , Crânio/patologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Células-Tronco/patologia , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/metabolismo , Sistema Nervoso Simpático/patologia , Fatores de Tempo , Proteínas de Peixe-Zebra/genética
19.
Development ; 140(11): 2354-64, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23615277

RESUMO

The zebrafish is a powerful genetic model that has only recently been used to dissect developmental pathways involved in oncogenesis. We hypothesized that operative pathways during embryogenesis would also be used for oncogenesis. In an effort to define RAS target genes during embryogenesis, gene expression was evaluated in Tg(hsp70-HRAS(G12V)) zebrafish embryos subjected to heat shock. dusp6 was activated by RAS, and this was used as the basis for a chemical genetic screen to identify small molecules that interfere with RAS signaling during embryogenesis. A KRAS(G12D)-induced zebrafish embryonal rhabdomyosarcoma was then used to assess the therapeutic effects of the small molecules. Two of these inhibitors, PD98059 and TPCK, had anti-tumor activity as single agents in both zebrafish embryonal rhabdomyosarcoma and a human cell line of rhabdomyosarcoma that harbored activated mutations in NRAS. PD98059 inhibited MEK1 whereas TPCK suppressed S6K1 activity; however, the combined treatment completely suppressed eIF4B phosphorylation and decreased translation initiation. Our work demonstrates that the activated pathways in RAS induction during embryogenesis are also important in oncogenesis and that inhibition of these pathways suppresses tumor growth.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Rabdomiossarcoma/patologia , Transdução de Sinais , Peixe-Zebra/embriologia , Proteínas ras/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular Tumoral , Fatores de Iniciação em Eucariotos/metabolismo , Flavonoides/farmacologia , Humanos , MAP Quinase Quinase 1/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Biossíntese de Proteínas , Rabdomiossarcoma/genética , Rabdomiossarcoma/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Tosilfenilalanil Clorometil Cetona/farmacologia , Transgenes , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
20.
Cancer Cell ; 21(5): 680-693, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22624717

RESUMO

Embryonal rhabdomyosarcoma (ERMS) is an aggressive pediatric sarcoma of muscle. Here, we show that ERMS-propagating potential is confined to myf5+ cells and can be visualized in live, fluorescent transgenic zebrafish. During early tumor growth, myf5+ ERMS cells reside adjacent normal muscle fibers. By late-stage ERMS, myf5+ cells are reorganized into distinct regions separated from differentiated tumor cells. Time-lapse imaging of late-stage ERMS revealed that myf5+ cells populate newly formed tumor only after seeding by highly migratory myogenin+ ERMS cells. Moreover, myogenin+ ERMS cells can enter the vasculature, whereas myf5+ ERMS-propagating cells do not. Our data suggest that non-tumor-propagating cells likely have important supportive roles in cancer progression and facilitate metastasis.


Assuntos
Movimento Celular , Rabdomiossarcoma Embrionário/patologia , Animais , Animais Geneticamente Modificados , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Progressão da Doença , Humanos , Camundongos , Camundongos SCID , Microscopia Confocal , Microscopia de Fluorescência por Excitação Multifotônica , Fator Regulador Miogênico 5/genética , Fator Regulador Miogênico 5/metabolismo , Miogenina/genética , Miogenina/metabolismo , Invasividade Neoplásica , Transplante de Neoplasias , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Proteínas Recombinantes de Fusão/metabolismo , Rabdomiossarcoma Embrionário/irrigação sanguínea , Rabdomiossarcoma Embrionário/genética , Rabdomiossarcoma Embrionário/metabolismo , Fatores de Tempo , Células Tumorais Cultivadas , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
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