RESUMEN
High hypoxia-inducible factor-2alpha (HIF-2alpha) protein levels predict poor outcome in neuroblastoma, and hypoxia dedifferentiates cultured neuroblastoma cells toward a neural crest-like phenotype. Here, we identify HIF-2alpha as a marker of normoxic neural crest-like neuroblastoma tumor-initiating/stem cells (TICs) isolated from patient bone marrows. Knockdown of HIF-2alpha reduced VEGF expression and induced partial sympathetic neuronal differentiation when these TICs were grown in vitro under stem cell-promoting conditions. Xenograft tumors of HIF-2alpha-silenced cells were widely necrotic, poorly vascularized, and resembled the bulk of tumor cells in clinical neuroblastomas by expressing additional sympathetic neuronal markers, whereas control tumors were immature, well-vascularized, and stroma-rich. Thus, HIF-2alpha maintains an undifferentiated state of neuroblastoma TICs. Because low differentiation is associated with poor outcome and angiogenesis is crucial for tumor growth, HIF-2alpha is an attractive target for neuroblastoma therapy.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular , Cresta Neural/metabolismo , Neuroblastoma/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Hipoxia de la Célula , Línea Celular Tumoral , Regulación hacia Abajo , Femenino , Humanos , Ratones , Ratones Desnudos , Factores de Crecimiento Endotelial Vascular/genética , Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Elevated expression of TLX (also called as NR2E1) in neuroblastoma (NB) correlates with unfavorable prognosis, and TLX is required for self-renewal of NB cells. Knockdown of TLX has been shown to reduce the NB sphere-forming ability. ASK1 (MAP3K5) and TLX expression are both enhanced in SP (side population) NB and patient-derived primary NB sphere cell lines, but the majority of non-SP NB lines express lower ASK1 expression. We found that ASK1 phosphorylated and stabilized TLX, which led induction of HIF-1α, and its downstream VEGF-A in an Akt dependent manner. In depleting ASK1 upon hypoxia, TLX decreased and the apoptosis ratio of NB cells was enhanced, while low-ASK1-expressing NB cell lines were refractory in TUNEL assay by using flow cytometry. Interestingly, primary NB spheres cell lines express only high levels of active pASK1Thr-838 but the established cell lines expressed inhibitory pASK1Ser-966, and both could be targeted by ASK1 depletion. We report a novel pro-survival role of ASK1 in the tumorigenic NB cell populations, which may be applied as a therapeutic target, inducing apoptosis specifically in cancer stem cells.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , MAP Quinasa Quinasa Quinasa 5/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patología , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Apoptosis/efectos de los fármacos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Bencimidazoles/metabolismo , Carbocianinas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Humanos , MAP Quinasa Quinasa Quinasa 5/química , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones SCID , Receptores Nucleares Huérfanos , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Dominios Proteicos , Inhibidores de Proteínas Quinasas/farmacología , Estabilidad Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The glial cell line-derived neurotrophic factor (GDNF) family of ligands play essential roles in promoting normal neural crest differentiation during embryogenesis, and, may have a therapeutic role in malignancies of neural crest origin, such as neuroblastoma. However, we report here that GDNF and neurturin blocked the growth inhibitory and neuritogenic effects of all-trans-retinoic acid in neuroblastoma cells in vitro. GDNF caused neuroblastoma cells to proliferate in the presence of a range of cytotoxic chemotherapeutic agents at low concentrations. Thus, our findings suggest a role for GDNF signaling in promoting resistance to differentiation or cytotoxic therapy of neuroblastoma, and, preclude their use in this neural crest tumor.
Asunto(s)
Antineoplásicos/antagonistas & inhibidores , Resistencia a Antineoplásicos/efectos de los fármacos , Factores Neurotróficos Derivados de la Línea Celular Glial/farmacología , Neuroblastoma/tratamiento farmacológico , Animales , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citotoxinas/antagonistas & inhibidores , Resistencia a Antineoplásicos/fisiología , Factor Neurotrófico Derivado de la Línea Celular Glial/farmacología , Inhibidores de Crecimiento/antagonistas & inhibidores , Inhibidores de Crecimiento/farmacología , Humanos , Ligandos , Ratones , Ratones Transgénicos , Neuroblastoma/metabolismo , Neuroblastoma/fisiopatología , Neurturina/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Tretinoina/antagonistas & inhibidores , Tretinoina/farmacologíaRESUMEN
Mass spectrometry-based technologies are increasingly utilized in drug discovery. Phosphoproteomics in particular has allowed for the efficient surveying of phosphotyrosine signaling pathways involved in various diseases states, most prominently in cancer. We describe a phosphotyrosine-based proteomics screening approach to identify signaling pathways and tyrosine kinase inhibitor targets in highly tumorigenic human lymphoma-like primary cells. We identified several receptor tyrosine kinase pathways and validated SRC family kinases (SFKs) as potential drug targets for targeted selection of small molecule inhibitors. BMS-354825 (dasatinib) and SKI-606 (bosutinib), second and third generation clinical SFK/ABL inhibitors, were found to be potent cytotoxic agents against tumorigenic cells with low toxicity to normal pediatric stem cells. Both SFK inhibitors reduced ERK1/2 and AKT phosphorylation and induced apoptosis. This study supports the adaptation of high-end mass spectrometry techniques for the efficient identification of candidate tyrosine kinases as novel therapeutic targets in primary cancer cell lines.
Asunto(s)
Compuestos de Anilina/farmacología , Nitrilos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Quinolinas/farmacología , Tiazoles/farmacología , Familia-src Quinasas/genética , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dasatinib , Perfilación de la Expresión Génica , Humanos , Linfoma , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , Proteómica , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Neuroblastoma (NB) is the most deadly extra-cranial solid tumour in children necessitating an urgent need for effective and less toxic treatments. One reason for the lack of efficacious treatments may be the inability of existing drugs to target the tumour-initiating or cancer stem cell population responsible for sustaining tumour growth, metastases and relapse. Here, we describe a strategy to identify compounds that selectively target patient-derived cancer stem cell-like tumour-initiating cells (TICs) while sparing normal paediatric stem cells (skin-derived precursors, SKPs) and characterize two therapeutic candidates. DECA-14 and rapamycin were identified as NB TIC-selective agents. Both compounds induced TIC death at nanomolar concentrations in vitro, significantly reduced NB xenograft tumour weight in vivo, and dramatically decreased self-renewal or tumour-initiation capacity in treated tumours. These results demonstrate that differential drug sensitivities between TICs and normal paediatric stem cells can be exploited to identify novel, patient-specific and potentially less toxic therapies.
Asunto(s)
Antibióticos Antineoplásicos/uso terapéutico , Decualinio/análogos & derivados , Células Madre Neoplásicas/efectos de los fármacos , Neuroblastoma/tratamiento farmacológico , Sirolimus/uso terapéutico , Bibliotecas de Moléculas Pequeñas/química , Animales , Apoptosis , Decualinio/química , Decualinio/uso terapéutico , Transporte de Electrón , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos NOD , Mitocondrias/genética , Mitocondrias/metabolismo , Células Madre Neoplásicas/metabolismo , Neuroblastoma/genética , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Neuroblastoma (NB) is an aggressive tumor of the developing peripheral nervous system that remains difficult to cure in the advanced stages. The poor prognosis for high-risk NB patients is associated with common disease recurrences that fail to respond to available therapies. NB tumor-initiating cells (TICs), isolated from metastases and primary tumors, may escape treatment and contribute to tumor relapse. New therapies that target the TICs may therefore prevent or treat tumor recurrences. EXPERIMENTAL DESIGN: We undertook a system-level characterization of NB TICs to identify potential drug targets against recurrent NB. We used next-generation RNA sequencing and/or human exon arrays to profile the transcriptomes of 11 NB TIC lines from six NB patients, revealing genes that are highly expressed in the TICs compared with normal neural crest-like cells and unrelated cancer tissues. We used gel-free two-dimensional liquid chromatography coupled to shotgun tandem mass spectrometry to confirm the presence of proteins corresponding to the most abundant TIC-enriched transcripts, thereby providing validation to the gene expression result. RESULTS: Our study revealed that genes in the BRCA1 signaling pathway are frequently misexpressed in NB TICs and implicated Aurora B kinase as a potential drug target for NB therapy. Treatment with a selective AURKB inhibitor was cytotoxic to NB TICs but not to the normal neural crest-like cells. CONCLUSION: This work provides the first high-resolution system-level analysis of the transcriptomes of 11 primary human NB TICs and identifies a set of candidate NB TIC-enriched transcripts for further development as therapeutic targets.
Asunto(s)
Antineoplásicos/uso terapéutico , Células Madre Neoplásicas/patología , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/patología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/fisiología , Aurora Quinasa B , Aurora Quinasas , Cromatografía Liquida/métodos , Evaluación Preclínica de Medicamentos , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Análisis por Micromatrices , Terapia Molecular Dirigida/métodos , Células Madre Neoplásicas/metabolismo , Neuroblastoma/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/farmacología , ARN Interferente Pequeño/uso terapéutico , Biología de Sistemas/métodos , Espectrometría de Masas en Tándem/métodos , Terapias en Investigación/métodos , Terapias en Investigación/tendencias , Estudios de Validación como AsuntoRESUMEN
The TrkA receptor tyrosine kinase is crucial for differentiation and survival of nerve-growth-factor-dependent neurons. Paradoxically, TrkA also induces cell death in pediatric tumor cells of neural origin, via an unknown mechanism. Here, we show that CCM2, a gene product associated with cerebral cavernous malformations, interacts with the juxtamembrane region of TrkA via its phosphotyrosine binding (PTB) domain and mediates TrkA-induced death in diverse cell types. Both the PTB and Karet domains of CCM2 are required for TrkA-dependent cell death, such that the PTB domain determines the specificity of the interaction, and the Karet domain links to death pathways. Downregulation of CCM2 in medulloblastoma or neuroblastoma cells attenuates TrkA-dependent death. Combined high expression levels of CCM2 and TrkA are correlated with long-term survival in a large cohort of human neuroblastoma patients. Thus, CCM2 is a key mediator of TrkA-dependent cell death in pediatric neuroblastic tumors.
Asunto(s)
Apoptosis/fisiología , Proteínas Portadoras/metabolismo , Proteínas de Microfilamentos/metabolismo , Neuroblastoma/fisiopatología , Receptor trkA/metabolismo , Animales , Proteínas Portadoras/genética , Muerte Celular/fisiología , Línea Celular , Células Cultivadas , Humanos , Meduloblastoma/fisiopatología , Ratones , Proteínas de Microfilamentos/genética , Mutación , Neuroblastoma/diagnóstico , Células PC12 , Pronóstico , Ratas , Receptor de Factor de Crecimiento Nervioso/metabolismo , Receptor trkA/genética , Receptor trkB/genética , Receptor trkB/metabolismoRESUMEN
Neuroblastoma is a heterogeneous pediatric tumor thought to arise from the embryonic neural crest. Identification of the cell responsible for propagating neuroblastomas is essential to understanding this often recurrent, rapidly progressing disease. We have isolated and characterized putative tumor-initiating cells from 16 tumors and bone marrow metastases from patients in all neuroblastoma risk groups. Dissociated cells from tumors or bone marrow grew as spheres in conditions used to culture neural crest stem cells, were capable of self-renewal, and exhibited chromosomal aberrations typical of neuroblastoma. Primary spheres from all tumor risk groups differentiated under neurogenic conditions to form neurons. Tumor spheres from low-risk tumors frequently formed large neuronal networks, whereas those from high-risk tumors rarely did. As few as 10 passaged tumor sphere cells from aggressive neuroblastoma injected orthotopically into severe combined immunodeficient/Beige mice formed large neuroblastoma tumors that metastasized to liver, spleen, contralateral adrenal and kidney, and lung. Furthermore, highly tumorigenic tumor spheres were isolated from the bone marrow of patients in clinical remission, suggesting that this population of cells may predict clinical behavior and serve as a biomarker for minimal residual disease in high-risk patients. Our data indicate that high-risk neuroblastoma contains a cell with cancer stem cell properties that is enriched in tumor-initiating capacity. These cells may serve as a model system to identify the molecular determinants of neuroblastoma and to develop new therapeutic strategies for this tumor.
Asunto(s)
Neoplasias de la Médula Ósea/patología , Neoplasias de la Médula Ósea/secundario , Diferenciación Celular , Células Madre Neoplásicas/patología , Neuroblastoma/patología , Animales , Células Cultivadas , Femenino , Citometría de Flujo , Humanos , Lactante , Ratones , Ratones SCID , FenotipoRESUMEN
The mechanisms causing persistence of embryonal cells that later give rise to tumors is unknown. One tumorigenic factor in the embryonal childhood tumor neuroblastoma is the MYCN protooncogene. Here we show that normal mice developed neuroblast hyperplasia in paravertebral ganglia at birth that completely regressed by 2 weeks of age. In contrast, ganglia from MYCN transgenic (TH-MYCN) mice demonstrated a marked increase in neuroblast hyperplasia and MycN expression during week 1. Regression of neuroblast hyperplasia was then delayed and incomplete before neuroblastoma tumor formation at 6 and 13 weeks in homo- and hemizygote mice, respectively. Paravertebral neuronal cells cultured from perinatal TH-MYCN mice exhibited 3- to 10-fold resistance to nerve growth factor (NGF) withdrawal, compared with normal mice. Both low- and high-affinity NGF receptors were expressed in perinatal neuroblast hyperplasia but not in neuroblastoma tumor tissue. MYCN transgene amplification was present at low levels in perinatal neuroblast hyperplasia from both homo- and hemizygote TH-MYCN mice. However, only in hemizygous mice did tumor formation correlate with a stepwise increase in the frequency of MYCN amplification. These data suggest that inappropriate perinatal MycN expression in paravertebral ganglia cells from TH-MYCN mice initiated tumorigenesis by altering the physiologic process of neural crest cell deletion. Persisting embryonal neural crest cells underwent further changes, such as MYCN amplification and repression of NGF receptor expression, during tumor progression. Our studies provide a model for studying perinatal factors influencing embryonal tumor initiation.