Olga Shakhova

Bmi1 is essential for cerebellar development and is overexpressed in human medulloblastomas

Overexpression of the polycomb group gene Bmi1 promotes cell proliferation and induces leukaemia through repression of Cdkn2a (also known as ink4a/Arf) tumour suppressors. Conversely, loss of Bmi1 leads to haematological defects and severe progressive neurological abnormalities in which de-repression of the ink4a/Arf locus is critically implicated. Here, we show that Bmi1 is strongly expressed in proliferating cerebellar precursor cells in mice and humans. Using Bmi1-null mice we demonstrate a crucial role for Bmi1 in clonal expansion of granule cell precursors both in vivo and in vitro. Deregulated proliferation of these progenitor cells, by activation of the sonic hedgehog (Shh) pathway, leads to medulloblastoma development. We also demonstrate linked overexpression of BMI1 and patched (PTCH), suggestive of SHH pathway activation, in a substantial fraction of primary human medulloblastomas. Together with the rapid induction of Bmi1 expression on addition of Shh or on overexpression of the Shh target Gli1 in cerebellar granule cell cultures, these findings implicate BMI1 overexpression as an alternative or additive mechanism in the pathogenesis of medulloblastomas, and highlight a role for Bmi1-containing polycomb complexes in proliferation of cerebellar precursor cells.

Sox10 promotes the formation and maintenance of giant congenital naevi and melanoma

Giant congenital naevi are pigmented childhood lesions that frequently lead to melanoma, the most aggressive skin cancer. The mechanisms underlying this malignancy are largely unknown, and there are no effective therapies. Here we describe a mouse model for giant congenital naevi and show that naevi and melanoma prominently express Sox10, a transcription factor crucial for the formation of melanocytes from the neural crest. Strikingly, Sox10 haploinsufficiency counteracts NrasQ61K-driven congenital naevus and melanoma formation without affecting the physiological functions of neural crest derivatives in the skin. Moreover, Sox10 is also crucial for the maintenance of neoplastic cells in vivo. In human patients, virtually all congenital naevi and melanomas are SOX10 positive. Furthermore, SOX10 silencing in human melanoma cells suppresses neural crest stem cell properties, counteracts proliferation and cell survival, and completely abolishes in vivo tumour formation. Thus, SOX10 represents a promising target for the treatment of congenital naevi and melanoma in human patients.

The epigenetic modifier EZH2 controls melanoma growth and metastasis through silencing of distinct tumour suppressors

Increased activity of the epigenetic modifier EZH2 has been associated with different cancers. However, evidence for a functional role of EZH2 in tumorigenesis in vivo remains poor, in particular in metastasizing solid cancers. Here we reveal central roles of EZH2 in promoting growth and metastasis of cutaneous melanoma. In a melanoma mouse model, conditional Ezh2 ablation as much as treatment with the preclinical EZH2 inhibitor GSK503 stabilizes the disease through inhibition of growth and virtually abolishes metastases formation without affecting normal melanocyte biology. Comparably, in human melanoma cells, EZH2 inactivation impairs proliferation and invasiveness, accompanied by re-expression of tumour suppressors connected to increased patient survival. These EZH2 target genes suppress either melanoma growth or metastasis in vivo, revealing the dual function of EZH2 in promoting tumour progression. Thus, EZH2-mediated epigenetic repression is highly relevant especially during advanced melanoma progression, which makes EZH2 a promising target for novel melanoma therapies.

Temporal control of neural crest lineage generation by Wnt/β-catenin signaling

Wnt/β-catenin signaling controls multiple steps of neural crest development, ranging from neural crest induction, lineage decisions, to differentiation. In mice, conditional β-catenin inactivation in premigratory neural crest cells abolishes both sensory neuron and melanocyte formation. Intriguingly, the generation of melanocytes is also prevented by activation of β-catenin in the premigratory neural crest, which promotes sensory neurogenesis at the expense of other neural crest derivatives. This raises the question of how Wnt/β-catenin signaling regulates the formation of distinct lineages from the neural crest. Using various Cre lines to conditionally activate β-catenin in neural crest cells at different developmental stages, we show that neural crest cell fate decisions in vivo are subject to temporal control by Wnt/β-catenin. Unlike in premigratory neural crest, β-catenin activation in migratory neural crest cells promotes the formation of ectopic melanoblasts, while the production of most other lineages is suppressed. Ectopic melanoblasts emerge at sites of neural crest target structures and in many tissues usually devoid of neural crest-derived cells. β-catenin activation at later stages in glial progenitors or in melanoblasts does not lead to surplus melanoblasts, indicating a narrow time window of Wnt/β-catenin responsiveness during neural crest cell migration. Thus, neural crest cells appear to be multipotent in vivo both before and after emigration from the neural tube but adapt their response to extracellular signals in a temporally controlled manner.

Cerebellar stem cells act as medulloblastoma-initiating cells in a mouse model and a neural stem cell signature characterizes a subset of human medulloblastomas

Cells with stem cell properties have been isolated from various areas of the postnatal mammalian brain, most recently from the postnatal mouse cerebellum. We show here that inactivation of the tumor suppressor genes Rb and p53 in these endogenous neural stem cells induced deregulated proliferation and resistance to apoptosis in vitro. Moreover, injection of these cells into mice formed medulloblastomas. Medulloblastomas are the most common malignant brain tumors of childhood, and despite recent advances in treatment they are associated with high morbidity and mortality. They are highly heterogeneous tumors characterized by a diverse genetic make-up and expression profile as well as variable prognosis. Here, we describe a novel ontogenetic pathway of medulloblastoma that significantly contributes to understanding their heterogeneity. Experimental medulloblastomas originating from neural stem cells preferentially expressed stem cell markers Nestin, Sox2 and Sox9, which were not expressed in medulloblastomas originating from granule-cell-restricted progenitors. Furthermore, the expression of these markers identified a subset of human medulloblastomas associated with a poorer clinical outcome.

Specific proteolytic cleavage of agrin regulates maturation of the neuromuscular junction

During the initial stage of neuromuscular junction (NMJ) formation, nerve-derived agrin cooperates with muscle-autonomous mechanisms in the organization and stabilization of a plaque-like postsynaptic specialization at the site of nerve–muscle contact. Subsequent NMJ maturation to the characteristic pretzel-like appearance requires extensive structural reorganization. We found that the progress of plaque-to-pretzel maturation is regulated by agrin. Excessive cleavage of agrin via transgenic overexpression of an agrin-cleaving protease, neurotrypsin, in motoneurons resulted in excessive reorganizational activity of the NMJs, leading to rapid dispersal of the synaptic specialization. By contrast, expression of cleavage-resistant agrin in motoneurons slowed down NMJ remodeling and delayed NMJ maturation. Neurotrypsin, which is the sole agrin-cleaving protease in the CNS, was excluded as the physiological agrin-cleaving protease at the NMJ, because NMJ maturation was normal in neurotrypsin-deficient mice. Together, our analyses characterize agrin cleavage at its proteolytic α- and β-sites by an as-yet-unspecified protease as a regulatory access for relieving the agrin-dependent constraint on endplate reorganization during NMJ maturation.

Mesenchymal stem cells and neural crest stem cells from adult bone marrow: characterization of their surprising similarities and differences.

The generation of neuronal cells from stem cells obtained from adult bone marrow is of significant clinical interest in order to design new cell therapy protocols for several neurological disorders. The recent identification in adult bone marrow of stem cells derived from the neural crest stem cells (NCSC) might explain the neuronal phenotypic plasticity shown by bone marrow cells. However, little information is available about the nature of these cells compared to mesenchymal stem cells (MSC), including their similarities and differences. In this paper, using transcriptomic as well as proteomic technologies, we compared NCSC to MSC and stromal nestin-positive cells, all of them isolated from adult bone marrow. We demonstrated that the nestin-positive cell population, which was the first to be described as able to differentiate into functional neurons, was a mixed population of NCSC and MSC. More interestingly, we demonstrated that MSC shared with NCSC the same ability to truly differentiate into Tuj1-positive cells when co-cultivated with paraformaldehyde-fixed cerebellar granule neurons. Altogether, those results suggest that both NCSC and MSC can be considered as important tools for cellular therapies in order to replace neurons in various neurological diseases.

Lack of Rb and p53 Delays Cerebellar Development and Predisposes to Large Cell Anaplastic Medulloblastoma through Amplification of N-Myc and Ptch2

Medulloblastomas are among the most common malignant brain tumors in childhood. They typically arise from neoplastic transformation of granule cell precursors in the cerebellum via deregulation of molecular pathways involved in normal cerebellar development. In a mouse model, we show here that impairment of the balance between proliferation and differentiation of granule cell precursors in the external granular layer of the developing cerebellum predisposes but is not sufficient to induce neoplastic transformation of these progenitor cells. Using array-based chromosomal comparative genomic hybridization, we show that genetic instability resulting from inactivation of the p53 pathway together with deregulation of proliferation induced by Rb loss eventually leads to neoplastic transformation of these cells by acquiring additional genetic mutations, mainly affecting N-Myc and Ptch2 genes. Moreover, we show that p53 loss influences molecular mechanisms that cannot be mimicked by the loss of either p19(ARF), p21, or ATM.

Conditional activation of Bmi1 expression regulates self-renewal, apoptosis, and differentiation of neural stem/progenitor cells in vitro and in vivo.

The Polycomb group protein Bmi1 is a key regulator of self‐renewal of embryonic and adult central nervous system stem cells, and its overexpression has been shown to occur in several types of brain tumors. In a Cre/LoxP‐based conditional transgenic mouse model, we show that fine‐tuning of Bmi1 expression in embryonic neural stem cell (NSC) is sufficient to increase their proliferation and self‐renewal potential both in vitro and in vivo. This is linked to downregulation of both the ink4a/ARF and the p21/Foxg1 axes. However, increased and ectopic proliferation induced by overexpression of Bmi1 in progenitors committed toward a neuronal lineage during embryonic cortical development, triggers apoptosis through a survivin‐mediated mechanism and leads to reduced brain size. Postnatally, however, increased self‐renewal capacity of neural stem/progenitor cells (NSPC) is independent of Foxg1 and resistance to apoptosis is observed in neural progenitors derived from NSC‐overexpressing Bmi1. Neoplastic transformation is absent in mice‐overexpressing Bmi1 aged up to 20 months. These studies provide strong evidence that fine tuning of Bmi1 expression is a viable tool to increase self‐renewal capacity of NSCs both in vitro and in vivo without eliciting neoplastic transformation of these cells. STEM Cells 2011;29:700–712

lncRNA Maturation to Initiate Heterochromatin Formation in the Nucleolus Is Required for Exit from Pluripotency in ESCs

The open chromatin of embryonic stem cells (ESCs) condenses into repressive heterochromatin as cells exit the pluripotent state. How the 3D genome organization is orchestrated and implicated in pluripotency and lineage specification is not understood. Here, we find that maturation of the long noncoding RNA (lncRNA) pRNA is required for establishment of heterochromatin at ribosomal RNA genes, the genetic component of nucleoli, and this process is inactivated in pluripotent ESCs. By using mature pRNA to tether heterochromatin at nucleoli of ESCs, we find that localized heterochromatin condensation of ribosomal RNA genes initiates establishment of highly condensed chromatin structures outside of the nucleolus. Moreover, we reveal that formation of such highly condensed, transcriptionally repressed heterochromatin promotes transcriptional activation of differentiation genes and loss of pluripotency. Our findings unravel the nucleolus as an active regulator of chromatin plasticity and pluripotency and challenge current views on heterochromatin regulation and function in ESCs.