Profiling cytotoxic microRNAs in pediatric and adult glioblastoma cells by high-content screening, identification, and validation of miR-1300.

MicroRNAs play an important role in the regulation of mRNA translation and have therapeutic potential in cancer and other diseases. To profile the landscape of microRNAs with significant cytotoxicity in the context of glioblastoma (GBM), we performed a high-throughput screen in adult and pediatric GBM cells using a synthetic oligonucleotide library representing all known human microRNAs. Bioinformatics analysis was used to refine this list and the top seven microRNAs were validated in a larger panel of GBM cells using state-of-the-art in vitro assays. The cytotoxic effect of our most relevant candidate was assessed in a preclinical model. Our screen identified ~100 significantly cytotoxic microRNAs with 70% concordance between cell lines. MicroRNA-1300 (miR-1300) was the most potent and robust candidate. We observed a striking binucleated phenotype in miR-1300 transfected cells due to cytokinesis failure followed by apoptosis. This was also observed in two stem-like patient-derived cultures. We identified the physiological role of miR-1300 as a regulator of endomitosis in megakaryocyte differentiation where blockade of cytokinesis is an essential step. In GBM cells, where miR-1300 is normally not expressed, the oncogene Epithelial Cell Transforming 2 (ECT2) was validated as a direct key target. ECT2 siRNA phenocopied the effects of miR-1300, and ECT2 overexpression led to rescue of miR-1300 induced binucleation. We showed that ectopic expression of miR-1300 led to decreased tumor growth in an orthotopic GBM model. Our screen provides a resource for the neuro-oncology community and identified miR-1300 as a novel regulator of endomitosis with translatable potential for therapeutic application.

Aggressive acute myeloid leukemia in PU.1/p53 double-mutant mice.

PU.1 downregulation within hematopoietic stem and progenitor cells (HSPCs) is the primary mechanism for the development of acute myeloid leukemia (AML) in mice with homozygous deletion of the upstream regulatory element (URE) of PU.1 gene. p53 is a well-known tumor suppressor that is often mutated in human hematologic malignancies including AML and adds to their aggressiveness; however, its genetic deletion does not cause AML in mouse. Deletion of p53 in the PU.1(ure/ure) mice (PU.1(ure/ure)p53(-/-)) results in more aggressive AML with shortened overall survival. PU.1(ure/ure)p53(-/-) progenitors express significantly lower PU.1 levels. In addition to URE deletion we searched for other mechanisms that in the absence of p53 contribute to decreased PU.1 levels in PU.1(ure/ure)p53(-/-) mice. We found involvement of Myb and miR-155 in downregulation of PU.1 in aggressive murine AML. Upon inhibition of either Myb or miR-155 in vitro the AML progenitors restore PU.1 levels and lose leukemic cell growth similarly to PU.1 rescue. The MYB/miR-155/PU.1 axis is a target of p53 and is activated early after p53 loss as indicated by transient p53 knockdown. Furthermore, deregulation of both MYB and miR-155 coupled with PU.1 downregulation was observed in human AML, suggesting that MYB/miR-155/PU.1 mechanism may be involved in the pathogenesis of AML and its aggressiveness characterized by p53 mutation.

5-azacitidine in aggressive myelodysplastic syndromes regulates chromatin structure at PU.1 gene and cell differentiation capacity.

Epigenetic 5-azacitidine (AZA) therapy of high-risk myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML) represents a promising, albeit not fully understood, approach. Hematopoietic transcription factor PU.1 is dynamically regulated by upstream regulatory element (URE), whose deletion causes downregulation of PU.1 leading to AML in mouse. In this study a significant group of the high-risk MDS patients, as well as MDS cell lines, displayed downregulation of PU.1 expression within CD34+ cells, which was associated with DNA methylation of the URE. AZA treatment in vitro significantly demethylated URE, leading to upregulation of PU.1 followed by derepression of its transcriptional targets and onset of myeloid differentiation. Addition of colony-stimulating factors (CSFs; granulocyte-CSF, granulocyte-macrophage-CSF and macrophage-CSF) modulated AZA-mediated effects on reprogramming of histone modifications at the URE and cell differentiation outcome. Our data collectively support the importance of modifying the URE chromatin structure as a regulatory mechanism of AZA-mediated activation of PU.1 and induction of the myeloid program in MDS.

The role of PU.1 and GATA-1 transcription factors during normal and leukemogenic hematopoiesis.

Hematopoiesis is coordinated by a complex regulatory network of transcription factors and among them PU.1 (Spi1, Sfpi1) represents a key molecule. This review summarizes the indispensable requirement of PU.1 during hematopoietic cell fate decisions and how the function of PU.1 can be modulated by protein-protein interactions with additional factors. The mutual negative regulation between PU.1 and GATA-1 is detailed within the context of normal and leukemogenic hematopoiesis and the concept of 'differentiation therapy' to restore normal cellular differentiation of leukemic cells is discussed.

Differential expression of Group I metabotropic glutamate receptors in motoneurons at low and high risk for degeneration in ALS.

Glutamate excitotoxicity has been suggested to play a role in amyotrophic lateral sclerosis (ALS), yet it remains unclear why some groups of motoneurons (MNs) are more vulnerable to degeneration than others. Our aim was to compare, in normal adult rats, the expression of Group I metabotropic glutamate receptors (mGluR1 and mGluR5) in MNs normally affected in ALS (XII and spinal MNs) with those which are spared (III and IV MNs). RT-PCR analysis of tissue punches taken from III and XII motor nuclei revealed mRNA for both 'a' and 'b' splice variants of the mGluR1 and mGluR5 receptor subtypes, with expression of the 'a' variant dominant for both receptor subtypes in III and XII nuclei. Immunolabeling for mGluR1a protein was strong in vulnerable (XII and spinal) but negligible in the resistant (III and IV) MNs. Immunoreactivity for mGluR5 was not detected in the cell bodies or proximal dendrites of any MN pool examined. Greater expression of mGluR1a receptor protein within vulnerable MN pools may predispose these neurons to neurodegeneration as seen in ALS.

GluR2 AMPA receptor subunit expression in motoneurons at low and high risk for degeneration in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder that results in selective degeneration of most, but not all, groups of motoneurons. The greater susceptibility of vulnerable motoneurons to glutamate excitotoxicity and neurodegeneration has been hypothesized to result from their lower expression of the GluR2 AMPA receptor subunit under control conditions, which renders these receptors permeable to calcium. To address the question of whether there is differential expression of the GluR2 subunit in motoneurons, we compared in normal adult rats expression of GluR2 mRNA and protein within two cranial motor nuclei that are either resistant (III; oculomotor nucleus) or vulnerable (XII; hypoglossal nucleus) to degeneration in ALS. RT-PCR analysis of tissue punched from III and XII motor nuclei detected mRNA for all AMPA subunits (GluR1-R4). In situ hybridization demonstrated no significant difference in GluR2 mRNA expression between III and XII nuclei. Immunohistochemical examination of GluR2 (and GluR4) protein levels demonstrated a similar pattern of the subunit expression in both motor nuclei. This equivalent expression of GluR2 mRNA and protein in motoneurons that differ in their vulnerability to degeneration in ALS suggests that reduced expression of GluR2 is not a factor predisposing motoneurons to degeneration.

Calcium binding proteins in motoneurons at low and high risk for degeneration in ALS.

Recent reports challenge the hypothesis that expression of calcium binding proteins contributes to the greater resistance of some motoneurons to degeneration in amyotrophic lateral sclerosis (ALS). We therefore re-examined, using immunohistochemistry, the expression of calbindin, calretinin and parvalbumin in vulnerable (hypoglossal, XII; and cervical spinal) and resistant (oculomotor, III) motoneurons of adult rats. Calbindin immunoreactivity was lacking in motor nuclei but strong in the dorsal horn. Calretinin was expressed in spinal, but not III or XII, motoneurons. Parvalbumin immunoreactivity, tested with a polyclonal antibody, was intense in spinal and III, but not XII, motoneurons; however, no staining in the ventral horn was observed with a monoclonal antibody. Differential expression of calretinin and parvalbumin within vulnerable motoneurons suggests that immunoreactivity for these proteins is not a reliable marker for resistance to degeneration in ALS.