RESUMEN
We identify a population of Protogenin-positive (PRTG+ve) MYChigh NESTINlow stem cells in the four-week-old human embryonic hindbrain that subsequently localizes to the ventricular zone of the rhombic lip (RLVZ). Oncogenic transformation of early Prtg+ve rhombic lip stem cells initiates group 3 medulloblastoma (Gr3-MB)-like tumors. PRTG+ve stem cells grow adjacent to a human-specific interposed vascular plexus in the RLVZ, a phenotype that is recapitulated in Gr3-MB but not in other types of medulloblastoma. Co-culture of Gr3-MB with endothelial cells promotes tumor stem cell growth, with the endothelial cells adopting an immature phenotype. Targeting the PRTGhigh compartment of Gr3-MB in vivo using either the diphtheria toxin system or chimeric antigen receptor T cells constitutes effective therapy. Human Gr3-MBs likely arise from early embryonic RLVZ PRTG+ve stem cells inhabiting a specific perivascular niche. Targeting the PRTGhigh compartment and/or the perivascular niche represents an approach to treat children with Gr3-MB.
RESUMEN
Medulloblastoma (MB) comprises a group of heterogeneous paediatric embryonal neoplasms of the hindbrain with strong links to early development of the hindbrain1-4. Mutations that activate Sonic hedgehog signalling lead to Sonic hedgehog MB in the upper rhombic lip (RL) granule cell lineage5-8. By contrast, mutations that activate WNT signalling lead to WNT MB in the lower RL9,10. However, little is known about the more commonly occurring group 4 (G4) MB, which is thought to arise in the unipolar brush cell lineage3,4. Here we demonstrate that somatic mutations that cause G4 MB converge on the core binding factor alpha (CBFA) complex and mutually exclusive alterations that affect CBFA2T2, CBFA2T3, PRDM6, UTX and OTX2. CBFA2T2 is expressed early in the progenitor cells of the cerebellar RL subventricular zone in Homo sapiens, and G4 MB transcriptionally resembles these progenitors but are stalled in developmental time. Knockdown of OTX2 in model systems relieves this differentiation blockade, which allows MB cells to spontaneously proceed along normal developmental differentiation trajectories. The specific nature of the split human RL, which is destined to generate most of the neurons in the human brain, and its high level of susceptible EOMES+KI67+ unipolar brush cell progenitor cells probably predisposes our species to the development of G4 MB.
Asunto(s)
Diferenciación Celular , Neoplasias Cerebelosas , Meduloblastoma , Metencéfalo , Diferenciación Celular/genética , Linaje de la Célula , Neoplasias Cerebelosas/clasificación , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/patología , Cerebelo/embriología , Cerebelo/patología , Subunidades alfa del Factor de Unión al Sitio Principal/genética , Proteínas Hedgehog/metabolismo , Histona Demetilasas , Humanos , Antígeno Ki-67/metabolismo , Meduloblastoma/clasificación , Meduloblastoma/genética , Meduloblastoma/patología , Metencéfalo/embriología , Metencéfalo/patología , Proteínas Musculares , Mutación , Factores de Transcripción Otx/deficiencia , Factores de Transcripción Otx/genética , Proteínas Represoras , Proteínas de Dominio T Box/metabolismo , Factores de TranscripciónRESUMEN
Messenger RNA is a flexible tool box that plays a key role in the dynamic regulation of gene expression. RNA modifications variegate the message conveyed by the mRNA. Similar to DNA and histone modifications, mRNA modifications are reversible and play a key role in the regulation of molecular events. Our understanding about the landscape of RNA modifications is still rudimentary in contrast to DNA and histone modifications. The major obstacle has been the lack of sensitive detection methods since they are non-editing events. However, with the advent of next-generation sequencing techniques, RNA modifications are being identified precisely at single nucleotide resolution. In recent years, methylation at the N6 position of adenine (m6 A) has gained the attention of RNA biologists. The m6 A modification has a set of writers (methylases), erasers (demethylases), and readers. Here, we provide a summary of interesting facts, conflicting findings, and recent advances in the technical and functional aspects of the m6 A epitranscriptome.
Asunto(s)
Adenosina/análogos & derivados , ARN Mensajero/genética , Adenosina/metabolismo , Animales , Línea Celular Tumoral , Regulación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Metiltransferasas/genética , Metiltransferasas/metabolismo , ARN Mensajero/metabolismoRESUMEN
Radiation is the standard therapy used for treating Glioblastoma (GBM), a grade IV brain cancer. Glioma Stem-like Cells (GSCs), an integral part of GBM, enforces resistance to radiation therapy of GBM. Studying the differential biomolecular composition of GSCs with varying levels of radiation sensitivity can aid in identifying the molecules and their associated pathways which impose resistance to cells thereby unraveling new targets which would serve as potential adjuvant therapy. Raman spectroscopy being a noninvasive, label free technique can determine the biomolecular constituent of cells under live conditions. In this study, we have deduced Raman spectral signatures to predict the radiosensitivity of any GSC accurately using the inherent and radiation induced biomolecular composition. Our study identified the differential regulation of several biomolecules which can be potential targets for adjuvant therapy. We radiosensitized the resistant GSCs using small molecule inhibitors specific to the metabolic pathways of these biomolecules. Efficient antitumor therapy can be attained with lower dosage of radiation along with these inhibitors and thus improving the survival rate of GBM patients with reduced side-effects from radiation.
Asunto(s)
Glioblastoma/diagnóstico , Células Madre Neoplásicas/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quimioterapia Adyuvante , Glioblastoma/terapia , Hemicolinio 3/química , Hemicolinio 3/farmacología , Humanos , Células Madre Neoplásicas/patología , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Espectrometría RamanRESUMEN
Despite recent advances in N6-methyladenosine (m6A) biology, the regulation of crucial RNA processing steps by the RNA methyltransferase-like 3 (METTL3) in glioma stem-like cells (GSCs) remains obscure. An integrated analysis of m6A-RIP (RNA immunoprecipitation) and total RNA-Seq of METTL3-silenced GSCs identified that m6A modification in GSCs is principally carried out by METTL3. The m6A-modified transcripts showed higher abundance compared to non-modified transcripts. Further, we showed that the METTL3 is essential for the expression of GSC-specific actively transcribed genes. Silencing METTL3 resulted in the elevation of several aberrant alternative splicing events. We also found that putative m6A reader proteins play a key role in the RNA stabilization function of METTL3. METTL3 altered A-to-I and C-to-U RNA editing events by differentially regulating RNA editing enzymes ADAR and APOBEC3A. Similar to protein-coding genes, lincRNAs (long intergenic non-coding RNAs) with m6A marks showed METTL3-dependent high expression. m6A modification of 3'UTRs appeared to result in a conformation-dependent hindrance to miRNA binding to their targets. The integrated analysis of the m6A regulome in METTL3-silenced GSCs showed global disruption in tumorigenic pathways that are indispensable for GSC maintenance and glioma progression. We conclude that METTL3 plays a vital role in many steps of RNA processing and orchestrates successful execution of oncogenic pathways in GSCs.
Asunto(s)
Adenosina/análogos & derivados , Glioma/genética , Metiltransferasas/genética , Transcripción Genética , Regiones no Traducidas 3'/genética , Adenosina/genética , Adenosina Desaminasa/genética , Carcinogénesis/genética , Línea Celular Tumoral , Citidina Desaminasa/genética , Regulación Neoplásica de la Expresión Génica , Glioma/patología , Histonas/genética , Humanos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Conformación de Ácido Nucleico , Proteínas/genética , Edición de ARN/genética , Empalme del ARN/genética , Proteínas de Unión al ARN/genética , Transducción de Señal/genéticaRESUMEN
The diffusely infiltrative nature of glioblastoma (GBM) makes them highly recurrent. IGF2 mRNA-binding protein 3 (IMP3), a GBM upregulated RNA binding protein, promotes glioma cell migration. An integrative bioinformatics analysis identified p65 (RELA), a subunit of NF-κB heterodimer as a target and an important mediator of IMP3 promoted glioma cell migration. IMP3 increased p65 protein levels without any change in p65 transcript levels, but promoted its polysome association. RIP-PCR demonstrated the binding of IMP3 to p65 transcript. UV crosslinking experiments with in vitro transcribed RNA confirmed the specific and direct binding of IMP3 to sites on p65 3'UTR. Further, IMP3 induced luciferase activity from p65 3'UTR reporter carrying wild type sites but not mutated sites. Exogenous overexpression of p65 from a 3'UTR-less construct rescued the reduced migration of glioma cells in IMP3 silenced condition. In addition, IMP3 silencing inhibited glioma stem-like cell maintenance and migration. The exogenous overexpression of 3'UTR-less p65 significantly alleviated the inhibition of neurosphere formation observed in IMP3 silenced glioma stem-like cells. Further, we show that IMP3 is transcriptionally activated by NF-κB pathway indicating the presence of a positive feedback loop between IMP3 and p65. This study establishes p65 as a novel target of IMP3 in increasing glioma cell migration and underscores the significance of IMP3-p65 feedback loop for therapeutic targeting in GBM.