Epigenetic modulation of a miR-296-5p:HMGA1 axis regulates Sox2 expression and glioblastoma stem cells.

Solid malignancies contain subsets of multipotent cells that grow as spheres and efficiently propagate tumors in xenograft models, reflecting a stem-like, self-renewing and tumor-propagating phenotype. These cancer 'stem cells (SCs)' have been shown to maintain tumor growth, contribute to resistance and drive tumor recurrence. Cancer cell stemness is dynamically influenced by epigenetic mechanisms and differentially regulated coding and noncoding RNAs. How these mechanisms specifically contribute to the generation and/or maintenance of cancer SCs remains unclear. This study identifies a novel epigenetically regulated circuit that integrates microRNA, chromatin remodeling and the reprogramming transcription factor Sox2 to regulate glioblastoma (GBM)-propagating SCs. We show that miR-296-5p expression is repressed in a DNA methylation-dependent manner under conditions that promote GBM cell stemness and that miR-296-5p inhibits GBM cell stemness and their capacity to self-renew as spheres and propagate glioma xenografts in vivo. We show that the chromatin remodeling protein HMGA1 functions as a downstream effector of these biological responses to miR-296-5p and regulates Sox2 expression, a master driver of cell stemness, by modifying chromatin architecture at the Sox2 promoter. These results show for the first time that miR-296-5p inhibits transcriptional mechanisms that support GBM SCs and identify a miR-296-5p:HMGA1:Sox2 axis as a novel regulator of GBM SCs and candidate pathway for targeting therapies directed at depleting tumors of their tumor-propagating stem cell subsets.

PAR1 inhibition suppresses the self-renewal and growth of A2B5-defined glioma progenitor cells and their derived gliomas in vivo.

Glioblastoma (GBM) remains the most common and lethal intracranial tumor. In a comparison of gene expression by A2B5-defined tumor-initiating progenitor cells (TPCs) to glial progenitor cells derived from normal adult human brain, we found that the F2R gene encoding PAR1 was differentially overexpressed by A2B5-sorted TPCs isolated from gliomas at all stages of malignant development. In this study, we asked if PAR1 is causally associated with glioma progression. Lentiviral knockdown of PAR1 inhibited the expansion and self-renewal of human GBM-derived A2B5(+) TPCs in vitro, while pharmacological inhibition of PAR 1 similarly slowed both the growth and migration of A2B5(+) TPCs in culture. In addition, PAR1 silencing potently suppressed tumor expansion in vivo, and significantly prolonged the survival of mice following intracranial transplantation of human TPCs. These data strongly suggest the importance of PAR1 to the self-renewal and tumorigenicity of A2B5-defined glioma TPCs; as such, the abrogation of PAR1-dependent signaling pathways may prove a promising strategy for gliomas.

DNMT-dependent suppression of microRNA regulates the induction of GBM tumor-propagating phenotype by Oct4 and Sox2.

Cancer stem-like cells represent poorly differentiated multipotent tumor-propagating cells that contribute disproportionately to therapeutic resistance and tumor recurrence. Transcriptional mechanisms that control the phenotypic conversion of tumor cells lacking tumor-propagating potential to tumor-propagating stem-like cells remain obscure. Here we show that the reprogramming transcription factors Oct4 and Sox2 induce glioblastoma cells to become stem-like and tumor-propagating via a mechanism involving direct DNA methyl transferase (DNMT) promoter transactivation, resulting in global DNA methylation- and DNMT-dependent downregulation of multiple microRNAs (miRNAs). We show that one such downregulated miRNA, miRNA-148a, inhibits glioblastoma cell stem-like properties and tumor-propagating potential. This study identifies a novel and targetable molecular circuit by which glioma cell stemness and tumor-propagating capacity are regulated.

Hypoxia-cultured human adipose-derived mesenchymal stem cells are non-oncogenic and have enhanced viability, motility, and tropism to brain cancer.

Adult human adipose-derived mesenchymal stem cells (hAMSCs) are multipotent cells, which are abundant, easily collected, and bypass the ethical concerns that plague embryonic stem cells. Their utility and accessibility have led to the rapid development of clinical investigations to explore their autologous and allogeneic cellular-based regenerative potential, tissue preservation capabilities, anti-inflammatory properties, and anticancer properties, among others. hAMSCs are typically cultured under ambient conditions with 21% oxygen. However, physiologically, hAMSCs exist in an environment of much lower oxygen tension. Furthermore, hAMSCs cultured in standard conditions have shown limited proliferative and migratory capabilities, as well as limited viability. This study investigated the effects hypoxic culture conditions have on primary intraoperatively derived hAMSCs. hAMSCs cultured under hypoxia (hAMSCs-H) remained multipotent, capable of differentiation into osteogenic, chondrogenic, and adipogenic lineages. In addition, hAMSCs-H grew faster and exhibited less cell death. Furthermore, hAMSCs-H had greater motility than normoxia-cultured hAMSCs and exhibited greater homing ability to glioblastoma (GBM) derived from brain tumor-initiating cells from our patients in vitro and in vivo. Importantly, hAMSCs-H did not transform into tumor-associated fibroblasts in vitro and were not tumorigenic in vivo. Rather, hAMSCs-H promoted the differentiation of brain cancer cells in vitro and in vivo. These findings suggest an alternative culturing technique that can enhance the function of hAMSCs, which may be necessary for their use in the treatment of various pathologies including stroke, myocardial infarction, amyotrophic lateral sclerosis, and GBM.

Regulation of glioblastoma stem cells by retinoic acid: role for Notch pathway inhibition.

It is necessary to understand mechanisms by which differentiating agents influence tumor-initiating cancer stem cells. Toward this end, we investigated the cellular and molecular responses of glioblastoma stem-like cells (GBM-SCs) to all-trans retinoic acid (RA). GBM-SCs were grown as non-adherent neurospheres in growth factor supplemented serum-free medium. RA treatment rapidly induced morphology changes, induced growth arrest at G1/G0 to S transition, decreased cyclin D1 expression and increased p27 expression. Immunofluorescence and western blot analysis indicated that RA induced the expression of lineage-specific differentiation markers Tuj1 and GFAP and reduced the expression of neural stem cell markers such as CD133, Msi-1, nestin and Sox-2. RA treatment dramatically decreased neurosphere-forming capacity, inhibited the ability of neurospheres to form colonies in soft agar and inhibited their capacity to propagate subcutaneous and intracranial xenografts. Expression microarray analysis identified ∼350 genes that were altered within 48 h of RA treatment. Affected pathways included retinoid signaling and metabolism, cell-cycle regulation, lineage determination, cell adhesion, cell-matrix interaction and cytoskeleton remodeling. Notch signaling was the most prominent of these RA-responsive pathways. Notch pathway downregulation was confirmed based on the downregulation of HES and HEY family members. Constitutive activation of Notch signaling with the Notch intracellular domain rescued GBM neurospheres from the RA-induced differentiation and stem cell depletion. Our findings identify mechanisms by which RA targets GBM-derived stem-like tumor-initiating cells and novel targets applicable to differentiation therapies for glioblastoma.

[Structural and functional characteristics of glutamate transporters: how they are related to epilepsy and oxidative stress]. / Caracteristicas estructurales y funcionales de los transportadores de glutamato: su relacion con la epilepsia y el estres oxidativo.

AIMS: The article highlights the general structural characteristics, functional properties and distribution of glutamate transporters, as well as the role they play in epilepsy and oxidative stress. DEVELOPMENT: Transporters of amino acids such as glutamate are considered to be proteins that are extremely important in the central nervous system because they participate in the capture of the neurotransmitter following its release in the synaptic cleft, thus putting an end to its effect and limiting glutamate-mediated excitability. These proteins belong to the family of Na+/K+ dependent transporters. A growing body of evidence has been gathered to show that these transporters are involved in several neuronal disorders, such as epilepsy and cerebral ischaemia. In this regard, it is considered that some defect in the structure of the transporters could affect their functioning and, therefore, favour the hyperexcitability produced by glutamate; this in turn would lead to the pathological disorders that are found in epilepsy. CONCLUSIONS: A detailed study of the structure and functioning of these transporters, as well as the role they play in the more common neurological diseases, such as epilepsy, would afford us a clearer view of new therapeutic alternatives with which to fight this kind of neuronal disorder in the future.

Características estructurales y funcionales de los transportadores de glutamato: su relación con la epilepsia y el estrés oxidativo / Structural and functional characteristics of glutamate transporters: how they are related to epilepsy and oxidative stress

Se enfatizan las características estructurales generales, las propiedades funcionales y la distribución de los transportadores de glutamato, así como la participación de éstos en la epilepsia y el estrés oxidativo. Desarrollo. Los transportadores de aminoácidos como el glutamato se consideran proteínas de suma importancia en el sistema nervioso central,ya que participan en la captura del neurotransmisor posterior a su liberación en la hendidura sináptica para finalizar de esta manera su efecto y limitar la excitabilidad mediada por el glutamato. Estas proteínas se incluyen en la familia de lostransportadores dependientes de Na+/K+. Numerosas evidencias demuestran la participación de los transportadores en variostrastornos neuronales, como la epilepsia y la isquemia cerebral. A este respecto se considera que algún defecto en la estructura de los transportadores podría afectar su función y, por tanto, favorecer la hiperexcitabilidad producida por el glutamato, de tal manera que conduzca a las alteraciones patológicas que se presentan en la epilepsia. Conclusiones. El estudio detalladode la estructura y función de estos transportadores, así como del papel que desempeñan en las enfermedades neurológicas más comunes como la epilepsia, permitirá visualizar con claridad nuevas alternativas terapéuticas para combatir este tipo de afecciones neuronales en el futuro

The article highlights the general structural characteristics, functional properties and distribution of glutamate transporters, as well as the role they play in epilepsy and oxidative stress. Development. Transporters of amino acids such as glutamate are considered to be proteins that are extremely important in the central nervous system because theyparticipate in the capture of the neurotransmitter following its release in the synaptic cleft, thus putting an end to its effect and limiting glutamate-mediated excitability. These proteins belong to the family of Na+/K+ dependent transporters. A growingbody of evidence has been gathered to show that these transporters are involved in several neuronal disorders, such as epilepsy and cerebral ischaemia. In this regard, it is considered that some defect in the structure of the transporters could affect their functioning and, therefore, favour the hyperexcitability produced by glutamate; this in turn would lead to thepathological disorders that are found in epilepsy. Conclusions. A detailed study of the structure and functioning of these transporters, as well as the role they play in the more common neurological diseases, such as epilepsy, would afford us a clearer view of new therapeutic alternatives with which to fight this kind of neuronal disorder in the future