Stemness and Cell Cycle Regulators and Their Modulation by Retinoic Acid in Ewing Sarcoma.

Retinoic acid (RA) regulates stemness and differentiation in human embryonic stem cells (ESCs). Ewing sarcoma (ES) is a pediatric tumor that may arise from the abnormal development of ESCs. Here we show that RA impairs the viability of SK-ES-1 ES cells and affects the cell cycle. Cells treated with RA showed increased levels of p21 and its encoding gene, CDKN1A. RA reduced mRNA and protein levels of SRY-box transcription factor 2 (SOX2) as well as mRNA levels of beta III Tubulin (TUBB3), whereas the levels of CD99 increased. Exposure to RA reduced the capability of SK-ES-1 to form tumorspheres with high expression of SOX2 and Nestin. Gene expression of CD99 and CDKN1A was reduced in ES tumors compared to non-tumoral tissue, whereas transcript levels of SOX2 were significantly higher in tumors. For NES and TUBB3, differences between tumors and control tissue did not reach statistical significance. Low expression of CD99 and NES, and high expression of SOX2, were significantly associated with a poorer patient prognosis indicated by shorter overall survival (OS). Our results indicate that RA may display rather complex modulatory effects on multiple target genes associated with the maintenance of stem cell's features versus their differentiation, cell cycle regulation, and patient prognosis in ES.

Gene Expression of GABAA Receptor Subunits and Association with Patient Survival in Glioma.

Rapid neuronal inhibition in the brain is mediated by γ-aminobutyric acid (GABA) activation of GABAA receptors. The GABRA5 gene, which encodes the α5 subunit of the GABAA receptor, has been implicated in an aggressive subgroup of medulloblastoma (MB), a type of pediatric brain tumor. However, the possible role of GABAA receptor subunits in glioma remains poorly understood. Here, we examined the expression of genes encoding GABAA receptor subunits in different types of glioma, and its possible association with patient prognosis assessed by overall survival (OS). Data were obtained from the French and The Cancer Genome Atlas Brain Lower Grade Glioma (TCGA-LGG) datasets and analyzed for expression of GABAA receptor subunit genes. OS was calculated using the Kaplan-Meier estimate. We found that genes GABRA2, GABRA3, GABRB3, GABRG1, and GABRG2 showed a significant association with OS, with higher gene expression indicating better prognosis. In patients with GBM, high expression of GABRA2 was associated with shorter OS, whereas, in contrast, higher levels of GABRB3 were associated with better prognosis indicated by longer OS. In patients with lower grade gliomas, GABRA3, GABRB3, GABRG1, and GABRG2, were associated with longer OS. High GABRB3 expression was related to longer survival when low grade glioma types were analyzed separately. Our results suggest an overall association between higher expression of most genes encoding GABAA receptor subunits and better prognosis in different types of glioma. Our findings support the possibility that down-regulation of GABAA receptors in glioma contributes to promoting tumor progression by reducing negative inhibition. These findings might contribute to further evaluation of GABAA receptors as a therapeutic target in glioma.

Gene Expression of CSF3R/CD114 Is Associated with Poorer Patient Survival in Glioma.

Gliomas comprise most cases of central nervous system (CNS) tumors. Gliomas afflict both adults and children, and glioblastoma (GBM) in adults represents the clinically most important type of malignant brain cancer, with a very poor prognosis. The cell surface glycoprotein CD114, which is encoded by the CSF3R gene, acts as the receptor for the granulocyte colony stimulating factor (GCSF), and is thus also called GCSFR or CSFR. CD114 is a marker of cancer stem cells (CSCs), and its expression has been reported in several cancer types. In addition, CD114 may represent one among various cases where brain tumors hijack molecular mechanisms involved in neuronal survival and synaptic plasticity. Here, we describe CSF3R mRNA expression in human gliomas and their association with patient prognosis as assessed by overall survival (OS). We found that the levels of CSF3R/CD114 transcripts are higher in a few different types of gliomas, namely astrocytoma, pilocytic astrocytoma, and GBM, in comparison to non-tumoral neural tissue. We also observed that higher expression of CSF3R/CD114 in gliomas is associated with poorer outcome as measured by a shorter OS. Our findings provide early evidence suggesting that CSF3R/CD114 shows a potential role as a prognosis marker of OS in patients with GBM.

Histone Methyltransferases G9a/Ehmt2 and GLP/Ehmt1 Are Associated with Cell Viability and Poorer Prognosis in Neuroblastoma and Ewing Sarcoma.

Changes in epigenetic programming have been proposed as being key events in the initiation and progression of childhood cancers. HMT euchromatic histone lysine methyltransferase 2 (G9a, EHMT2), which is encoded by the G9a (Ehmt2) gene, as well as its related protein GLP, which is encoded by the GLP/Ehmt1 gene, participate in epigenetic regulation by contributing to a transcriptionally repressed chromatin state. G9a/GLP activation has been reported in several cancer types. Herein, we evaluated the role of G9a in two solid pediatric tumors: neuroblastoma (NB) and Ewing sarcoma (ES). Our results show that G9a/Ehmt2 and GLP/Ehmt1 expression is higher in tumors with poorer prognosis, including St4 International Neuroblastoma Staging System (INSS) stage, MYCN amplified NB, and metastatic ES. Importantly, higher G9a and GLP levels were associated with shorter patient overall survival (OS) in both NB and ES. Moreover, pharmacological inhibition of G9a/GLP reduced cell viability in NB and ES cells. These findings suggest that G9a and GLP are associated with more aggressive NB and ES tumors and should be further investigated as being epigenetic targets in pediatric solid cancers.

ZEB1 is a Subgroup-Specific Marker of Prognosis and Potential Drug Target in Medulloblastoma.

Medulloblastoma (MB) is a malignant brain tumor that afflicts mostly children and adolescents and presents four distinct molecular subgroups, known as WNT, SHH, Group 3, and Group 4. ZEB1 is a transcription factor that promotes the expression of mesenchymal markers while restraining expression of epithelial and polarity genes. Because of ZEB1 involvement in cerebellum development, here we investigated the role of ZEB1 in MB. We found increased expression of ZEB1 in MB tumor samples compared to normal cerebellar tissue. Expression was higher in the SHH subgroup when compared to all other MB molecular subgroups. High ZEB1 expression was associated with poor prognosis in Group 3 and Group 4, whereas in patients with WNT tumors poorer prognosis were related to lower ZEB1 expression. There was a moderate correlation between ZEB1 and MYC expression in Group 3 and Group 4 MB. Treatment with the immunomodulator and histone deacetylase (HDAC) inhibitor fingolimod (FTY720) reduced ZEB1 expression specifically in D283 cells, which are representative of Group 3 and Group 4 MB. These findings reveal novel subgroup-specific associations of ZEB1 expression with survival in patients with MB and suggest that ZEB1 expression can be reduced by pharmacological agents that target HDAC activity.

Reverse Engineering of Ewing Sarcoma Regulatory Network Uncovers PAX7 and RUNX3 as Master Regulators Associated with Good Prognosis.

Ewing Sarcoma (ES) is a rare malignant tumor occurring most frequently in adolescents and young adults. The ES hallmark is a chromosomal translocation between the chromosomes 11 and 22 that results in an aberrant transcription factor (TF) through the fusion of genes from the FET and ETS families, commonly EWSR1 and FLI1. The regulatory mechanisms behind the ES transcriptional alterations remain poorly understood. Here, we reconstruct the ES regulatory network using public available transcriptional data. Seven TFs were identified as potential MRs and clustered into two groups: one composed by PAX7 and RUNX3, and another composed by ARNT2, CREB3L1, GLI3, MEF2C, and PBX3. The MRs within each cluster act as reciprocal agonists regarding the regulation of shared genes, regulon activity, and implications in clinical outcome, while the clusters counteract each other. The regulons of all the seven MRs were differentially methylated. PAX7 and RUNX3 regulon activity were associated with good prognosis while ARNT2, CREB3L1, GLI3, and PBX3 were associated with bad prognosis. PAX7 and RUNX3 appear as highly expressed in ES biopsies and ES cell lines. This work contributes to the understanding of the ES regulome, identifying candidate MRs, analyzing their methilome and pointing to potential prognostic factors.