RESUMEN
Medulloblastoma is one of the most common malignant brain tumors of children, and 30% of medulloblastomas are driven by gain-of-function genetic lesions in the Sonic Hedgehog (SHH) signaling pathway. EYA1, a haloacid dehalogenase phosphatase and transcription factor, is critical for tumorigenesis and proliferation of SHH medulloblastoma (SHH-MB). Benzarone and benzbromarone have been identified as allosteric inhibitors of EYA proteins. Using benzarone as a point of departure, we developed a panel of 35 derivatives and tested them in SHH-MB. Among these compounds, DS-1-38 functioned as an EYA antagonist and opposed SHH signaling. DS-1-38 inhibited SHH-MB growth in vitro and in vivo, showed excellent brain penetrance, and increased the lifespan of genetically engineered mice predisposed to fatal SHH-MB. These data suggest that EYA inhibitors represent promising therapies for pediatric SHH-MB. SIGNIFICANCE: Development of a benzarone derivative that inhibits EYA1 and impedes the growth of SHH medulloblastoma provides an avenue for improving treatment of this malignant pediatric brain cancer.
Asunto(s)
Benzbromarona/análogos & derivados , Neoplasias Encefálicas , Neoplasias Cerebelosas , Meduloblastoma , Animales , Ratones , Humanos , Niño , Proteínas Hedgehog , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/genética , Neoplasias Cerebelosas/tratamiento farmacológicoRESUMEN
Many genes that drive normal cellular development also contribute to oncogenesis. Medulloblastoma (MB) tumors likely arise from neuronal progenitors in the cerebellum, and we hypothesized that the heterogeneity observed in MBs with sonic hedgehog (SHH) activation could be due to differences in developmental pathways. To investigate this question, here we perform single-nucleus RNA sequencing on highly differentiated SHH MBs with extensively nodular histology and observed malignant cells resembling each stage of canonical granule neuron development. Through innovative computational approaches, we connect these results to published datasets and find that some established molecular subtypes of SHH MB appear arrested at different developmental stages. Additionally, using multiplexed proteomic imaging and MALDI imaging mass spectrometry, we identify distinct histological and metabolic profiles for highly differentiated tumors. Our approaches are applicable to understanding the interplay between heterogeneity and differentiation in other cancers and can provide important insights for the design of targeted therapies.
Asunto(s)
Neoplasias Cerebelosas , Meduloblastoma , Humanos , Proteínas Hedgehog/genética , Meduloblastoma/genética , Proteómica , Cerebelo , Neoplasias Cerebelosas/genéticaRESUMEN
Sonic hedgehog signaling regulates processes of embryonic development across multiple tissues, yet factors regulating context-specific Shh signaling remain poorly understood. Exome sequencing of families with polymicrogyria (disordered cortical folding) revealed multiple individuals with biallelic deleterious variants in TMEM161B, which encodes a multi-pass transmembrane protein of unknown function. Tmem161b null mice demonstrated holoprosencephaly, craniofacial midline defects, eye defects, and spinal cord patterning changes consistent with impaired Shh signaling, but were without limb defects, suggesting a CNS-specific role of Tmem161b. Tmem161b depletion impaired the response to Smoothened activation in vitro and disrupted cortical histogenesis in vivo in both mouse and ferret models, including leading to abnormal gyration in the ferret model. Tmem161b localizes non-exclusively to the primary cilium, and scanning electron microscopy revealed shortened, dysmorphic, and ballooned ventricular zone cilia in the Tmem161b null mouse, suggesting that the Shh-related phenotypes may reflect ciliary dysfunction. Our data identify TMEM161B as a regulator of cerebral cortical gyration, as involved in primary ciliary structure, as a regulator of Shh signaling, and further implicate Shh signaling in human gyral development.
Asunto(s)
Hurones , Proteínas Hedgehog , Animales , Femenino , Humanos , Ratones , Embarazo , Sistema Nervioso Central/metabolismo , Cilios/genética , Cilios/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Ratones Noqueados , Transducción de SeñalRESUMEN
Hallmark mutations in WNT and SHH medulloblastoma are usually distinct, but DDX3X is often mutated in both subgroups. In this issue of Developmental Cell, Patmore et al. identify Ddx3x as essential for hindbrain patterning, cell fate determination, and as a tumor suppressor gene that restricts cell lineage progression in tumorigenesis.
Asunto(s)
Neoplasias Cerebelosas , Meduloblastoma , Carcinogénesis , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Humanos , Meduloblastoma/genética , Mutación , Rombencéfalo/metabolismoRESUMEN
During neural development, stem and precursor cells can divide either symmetrically or asymmetrically. The transition between symmetric and asymmetric cell divisions is a major determinant of precursor cell expansion and neural differentiation, but the underlying mechanisms that regulate this transition are not well understood. Here, we identify the Sonic hedgehog (Shh) pathway as a critical determinant regulating the mode of division of cerebellar granule cell precursors (GCPs). Using partial gain and loss of function mutations within the Shh pathway, we show that pathway activation determines spindle orientation of GCPs, and that mitotic spindle orientation correlates with the mode of division. Mechanistically, we show that the phosphatase Eya1 is essential for implementing Shh-dependent GCP spindle orientation. We identify atypical protein kinase C (aPKC) as a direct target of Eya1 activity and show that Eya1 dephosphorylates a critical threonine (T410) in the activation loop. Thus, Eya1 inactivates aPKC, resulting in reduced phosphorylation of Numb and other components that regulate the mode of division. This Eya1-dependent cascade is critical in linking spindle orientation, cell cycle exit and terminal differentiation. Together these findings demonstrate that a Shh-Eya1 regulatory axis selectively promotes symmetric cell divisions during cerebellar development by coordinating spindle orientation and cell fate determinants.
Asunto(s)
División Celular/fisiología , Cerebelo/metabolismo , Proteínas Hedgehog/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células-Madre Neurales/metabolismo , Neurogénesis/fisiología , Proteínas Nucleares/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Animales , Cerebelo/embriología , Cerebelo/crecimiento & desarrollo , Ratones , Ratones Mutantes , Células-Madre Neurales/citología , Transducción de Señal/fisiologíaRESUMEN
Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries. In comparison, prophase spermatocytes can be collected from juvenile male mice with higher yields without the need for microdissection. However, it is difficult to obtain a pure synchronized population of cells at specific stages due to the heterogeneity of meiotic and post-meiotic germ cell populations in the juvenile and adult testis. For later stages of meiosis, it is advantageous to assess oocytes undergoing meiosis I (MI) or meiosis II (MII), because groups of mature oocytes can be collected from adult female mice and stimulated to resume meiosis in culture. Here, methods for meiotic chromatin spread preparations using oocytes dissected from fetal, neonatal and adult ovaries are described with accompanying video demonstrations. Chromosome missegregation events in mammalian oocytes are frequent, particularly during MI. These techniques can be used to assess and characterize the effects of different mutations or environmental exposures during various stages of oogenesis. As there are distinct differences between oogenesis and spermatogenesis, the techniques described within are invaluable to increase our understanding of mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.
Asunto(s)
Cromatina/metabolismo , Metafase/genética , Oocitos/metabolismo , Profase/genética , Animales , Femenino , Ratones , EmbarazoRESUMEN
Multiple aspects of Drosophila oogenesis, including germline stem cell activity, germ cell differentiation, and follicle survival, are regulated by the steroid hormone ecdysone. While the transcriptional targets of ecdysone signaling during development have been studied extensively, targets in the ovary remain largely unknown. Early studies of salivary gland polytene chromosomes led to a model in which ecdysone stimulates a hierarchical transcriptional cascade, wherein a core group of ecdysone-sensitive transcription factors induce tissue-specific responses by activating secondary branches of transcriptional targets. More recently, genome-wide approaches have identified hundreds of putative ecdysone-responsive targets. Determining whether these putative targets represent bona fide targets in vivo, however, requires that they be tested via traditional mutant analysis in a cell-type specific fashion. To investigate the molecular mechanisms whereby ecdysone signaling regulates oogenesis, we used genetic mosaic analysis to screen putative ecdysone-responsive genes for novel roles in the control of the earliest steps of oogenesis. We identified a cohort of genes required for stem cell maintenance, stem and progenitor cell proliferation, and follicle encapsulation, growth, and survival. These genes encode transcription factors, chromatin modulators, and factors required for RNA transport, stability, and ribosome biogenesis, suggesting that ecdysone might control a wide range of molecular processes during oogenesis. Our results suggest that, although ecdysone target genes are known to have cell type-specific roles, many ecdysone response genes that control larval or pupal cell types at developmental transitions are used reiteratively in the adult ovary. These results provide novel insights into the molecular mechanisms by which ecdysone signaling controls oogenesis, laying new ground for future studies.
Asunto(s)
Drosophila melanogaster/genética , Ecdisona/metabolismo , Regulación del Desarrollo de la Expresión Génica , Oogénesis/genética , Ovario/fisiología , Animales , Animales Modificados Genéticamente , Linaje de la Célula , Supervivencia Celular/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Ecdisona/farmacología , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Técnicas Genéticas , Ribonucleoproteínas Nucleares Heterogéneas/genética , Larva/efectos de los fármacos , Larva/genética , Mosaicismo , Mutación , Oogénesis/efectos de los fármacos , Ovario/citología , Pupa/genética , Receptores de Esteroides/genética , Células Madre/fisiologíaRESUMEN
The Smc5/6 complex, along with cohesin and condensin, is a member of the structural maintenance of chromosome (SMC) family, large ring-like protein complexes that are essential for chromatin structure and function. Thanks to numerous studies of the mitotic cell cycle, Smc5/6 has been implicated to have roles in homologous recombination, restart of stalled replication forks, maintenance of ribosomal DNA (rDNA) and heterochromatin, telomerase-independent telomere elongation, and regulation of chromosome topology. The nature of these functions implies that the Smc5/6 complex also contributes to the profound chromatin changes, including meiotic recombination, that characterize meiosis. Only recently, studies in diverse model organisms have focused on the potential meiotic roles of the Smc5/6 complex. Indeed, Smc5/6 appears to be essential for meiotic recombination. However, due to both the complexity of the process of meiosis and the versatility of the Smc5/6 complex, many additional meiotic functions have been described. In this review, we provide a clear overview of the multiple functions found so far for the Smc5/6 complex in meiosis. Additionally, we compare these meiotic functions with the known mitotic functions in an attempt to find a common denominator and thereby create clarity in the field of Smc5/6 research.