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Glioma stem cells (GSCs) contribute to therapy resistance and poor outcomes for glioma patients. A significant feature of GSCs is their ability to grow in an acidic microenvironment. However, the mechanism underlying the rewiring of their metabolism in low pH remains elusive. Here, using metabolomics and metabolic flux approaches, we cultured GSCs at pH 6.8 and pH 7.4 and found that cells cultured in low pH exhibited increased de novo purine nucleotide biosynthesis activity. The overexpression of glucose-6-phosphate dehydrogenase, encoded by G6PD or H6PD, supports the metabolic dependency of GSCs on nucleotides when cultured under acidic conditions, by enhancing the pentose phosphate pathway (PPP). The high level of reduced glutathione (GSH) under acidic conditions also causes demand for the PPP to provide NADPH. Taken together, upregulation of G6PD/H6PD in the PPP plays an important role in acidic-driven purine metabolic reprogramming and confers a predilection toward glioma progression. Our findings indicate that targeting G6PD/H6PD, which are closely related to glioma patient survival, may serve as a promising therapeutic target for improved glioblastoma therapeutics. An integrated metabolomics and metabolic flux analysis, as well as considering microenvironment and cancer stem cells, provide a precise insight into understanding cancer metabolic reprogramming.

Acidosis is a significant feature of the tumor microenvironment in glioma, and it is closely related to multiple biological functions of cancer stem cells. Here, we found that the self-renewal ability, the mitochondrial activity and ATP production were elevated in stem cell-like glioma cells (SLCs) under acidic microenvironment, which promoted and maintained the stemness of SLCs. Under acidosis, 25-hydroxy vitamin D 3 -24-hydroxylase (CYP24A1) was upregulated and catalyzed the fast degradation of 1α,25(OH) 2 D 3 . We further revealed that the active form of vitamin D (1α,25(OH) 2 D 3 ) could inhibit the expression of stemness markers, attenuate acidosis-induced increase of self-renewal ability and mitochondrial respiration in stem cell-like glioma cells. Our study indicates that the acidosis-CYP24A1-vitamin D pathway may be a key regulator of the cancer stem cell phenotype in malignant glioma and point out the potential value for the utilization of vitamin D to target cancer stem cells and to restrain the growth of malignant glioma in the future.

Glioma stem cells (GSCs) are a subset of tumor cells that drive glioma initiation and progression. The molecular mechanisms underlying the maintenance of GSCs are still poorly understood. Here we investigated the role of Rho GDP dissociation inhibitor α (RhoGDIα) in GSCs. RhoGDIα was down-regulated in glioma stem cells. Over-expression of RhoGDIα suppressed the self-renewal and tumorigenesis of GSCs. Further data showed that RhoGDIα inhibited the transcription activity of stem cell marker Oct4. Moreover, inactivation of ROCK1, a downstream effector of RhoGDIα, also decreased the self-renewal and Oct4 transcription activity, and rescued the effects caused by RhoGDIα knockdown. Our results indicate that RhoGDIα is involved in the maintenance of GSCs., Competing Interests: The authors declare no conflicts of interest.

RNA-RNA and protein-RNA interactions are essential for post-transcriptional regulation in normal development and may be deregulated in cancer initiation and progression. The RNA-binding protein PCBP2, an oncogenic protein in human malignant gliomas, is an essential regulator of mRNA and miRNA biogenesis, stability and activity. Here, we identified Rho GDP dissociation inhibitor α (ARHGDIA) as a target mRNA that binds to PCBP2, and we uncovered the role of ARHGDIA as a putative metastasis suppressor through analyses of in vitro and in vivo models of EMT and metastasis. Furthermore, we demonstrated that ARHGDIA is a potential target of miR-151-5p and miR-16 in gliomas. The interaction between PCBP2 and the 3'UTR of the ARHGDIA mRNA may induce a local change in RNA structure that favors subsequent binding of miR-151-5p and miR-16, thus leading to the suppression of ARHGDIA expression. PCBP2 may facilitate miR-151-5p and miR-16 promotion of glioma cell migration and invasion through mitigating the function of ARHGDIA.

Brain-enriched miR-128 is repressed in glioma cells, and could inhibit the proliferation of gliomas by targeting genes such as E2F3a and BMI1. To identify more targets of miR-128 in glioblastoma multiforme, the pulse stable isotope labeling with amino acids in cell culture (pSILAC) technique was used to test its impact on whole protein synthesis in T98G glioma cells. We successfully identified 1897 proteins, of which 1459 proteins were quantified. Among them, 133 proteins were downregulated after the overexpression of miR-128. Through predictions using various bioinformatics tools, 13 candidate target genes were chosen. A luciferase assay validated that 11 of 13 selected genes were potential targets of miR-128, and a mutagenesis experiment confirmed CBFB, CORO1C, GLTP, HnRNPF, and TROVE2 as the target genes. Moreover, we observed that the expression of CORO1C, TROVE2, and HnRNPF were higher in glioma cell lines compared to normal brain tissues and presented a tendency toward downregulation after overexpression of miR-128 in T98G cells. Furthermore, we have validated that CORO1C, TROVE2, and HnRNPF could inhibit glioma cell proliferation. In sum, our data showed that the integration of pSILAC and bioinformatics analysis was an efficient method for seeking the targets of miRNAs, and plentiful targets of miR-128 were screened and laid the foundation for research into the miR-128 regulation network., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Although the roles of circadian Clock genes and microRNAs in tumorigenesis have been profoundly studied, mechanisms of cross-talk between them in regulation of gliomagenesis are poorly understood. Here we show that the expression level of CLOCK is significantly increased in high-grade human glioma tissues and glioblastoma cell lines. In contrast miR-124 is attenuated in similar samples. Further studies show that Clock is a direct target of miR-124, and either restoration of miR-124 or silencing of CLOCK can reduce the activation of NF-κB. In conclusion, we suggest that as a target of glioma suppressor miR-124, CLOCK positively regulates glioma proliferation and migration by reinforcing NF-κB activity., (Copyright © 2013. Published by Elsevier B.V.)

PCBP2 is a member of the poly(C)-binding protein (PCBP) family, which plays an important role in posttranscriptional and translational regulation by interacting with single-stranded poly(C) motifs in target mRNAs. Several PCBP family members have been reported to be involved in human malignancies. Here, we show that PCBP2 is upregulated in human glioma tissues and cell lines. Knockdown of PCBP2 inhibited glioma growth in vitro and in vivo through inhibition of cell-cycle progression and induction of caspase-3-mediated apoptosis. Thirty-five mRNAs were identified as putative PCBP2 targets/interactors using RIP-ChIP protein-RNA interaction arrays in a human glioma cell line, T98G. Four-and-a-half LIM domain 3 (FHL3) mRNA was downregulated in human gliomas and was identified as a PCBP2 target. Knockdown of PCBP2 enhanced the expression of FHL3 by stabilizing its mRNA. Overexpression of FHL3 attenuated cell growth and induced apoptosis. This study establishes a link between PCBP2 and FHL3 proteins and identifies a new pathway for regulating glioma progression.

It has been reported that ubiquitin-conjugating enzyme 9 (Ubc9), the unique enzyme2 in the sumoylation pathway, is up-regulated in many cancers. However, the expression and regulation of UBC9 in glioma remains unknown. In this study, we found that Ubc9 was up-regulated in glioma tissues and cell lines compared to a normal control. UBC9 knockdown by small interfering RNA (siRNA) affected cell proliferation and apoptosis in T98G cells. Further experiments revealed that microRNA (miR)-214 directly targeted the 3' untranslated region (UTR) of UBC9 and that there was an inverse relationship between the expression levels of miR-214 and UBC9 protein in glioma tissues and cells. miR-214 overexpression suppressed the endogenous UBC9 protein and affected T98G cell proliferation. These findings suggest that miR-214 reduction facilitates UBC9 expression and is involved in the regulation of glioma cell proliferation.

Migration-proliferation dichotomy is a common mechanism in gliomagenesis; however, an understanding of the exact molecular mechanism of this "go or grow" phenomenon remains largely incomplete. In the present study, we first found that microRNA-9 (miR-9) is highly expressed in glioma cells. MiR-9 inhibited the proliferation and promoted the migration of glioma cells by directly targeting cyclic AMP response element-binding protein (CREB) and neurofibromin 1 (NF1), respectively. Our data also suggested a migration-inhibitory role for CREB through directly regulating the transcription of NF1. Furthermore, we found that the transcription of miR-9-1 is under CREB's control, forming a negative feedback minicircuitry. Taken together, miR-9 inhibits proliferation but promotes migration, whereas CREB plays a pro-proliferative and anti-migratory role, suggesting that the CREB-miR-9 negative feedback minicircuitry plays a critical role in the determination of "go or grow" in glioma cells.

Here we report the negative correlation of phosphorylation of Smad1 and BMPR-IB expression with the development of human glioma. Western blot analysis showed that expression of both phospho-Smad1/5/8 and BMPR-IB were decreased in malignant glioma tissues compared with normal brain tissues. Kaplan-Meier survival curves revealed that lower expression ratio of phospho-Smad1/5/8 to Smad1 expression significantly correlates with poor patient survival. Transient transfection of BMPR-IB activates Smad1 signaling and induces differentiation and apoptosis of U251 and U87 glioblastoma cells. The effects could be blocked by cotransfection of Smad6. These results might provide new molecular marker and target for glioma diagnosis and therapy.

Nectin-like molecule 1 (NECL1)/CADM3/IGSF4B/TSLL1/SynCAM3 is a neural tissue-specific immunoglobulin-like cell-cell adhesion molecule downregulated at the mRNA level in 12 human glioma cell lines. Here we found that the expression of NECL1 was lost in six glioma cell lines and 15 primary glioma tissues at both RNA and protein levels. Re-expression of NECL1 into glioma cell line U251 would repress cell proliferation in vitro by inducing cell cycle arrest. And also NECL1 could decrease the growth rate of tumors in nude mice in vivo. To further investigate the mechanism why NECL1 was silenced in glioma, the basic promoter region located at -271 to +81 in NECL1 genomic sequence was determined. DNA bisulfite sequencing was performed to study the methylation status of CpG islands in NECL1 promoter; however, no hypermethylated CpG site was found. Additionally, the activity of histone deacetylase (HDACs) in glioma was higher than that in normal brain tissues, and the expression of NECL1 in glioma cell lines could be reactivated by HDACs inhibitor-Trichostatin A (TSA). So the loss of NECL1 in glioma was at least partly caused by histone deacetylation. Luciferase reporter assays, chromatin immunoprecipitation and co-immunoprecipitation (co-IP) assays indicated that Sp1 played an important role in this process by binding to either HDAC1 in untreated glioma cells or p300/CBP in TSA treated cells. Our finding suggests that NECL1 may act as a tumor suppressor in glioma and loss of it in glioma may be caused by histone deacetylation., ((c) 2008 Wiley-Liss, Inc.)

MicroRNAs have been linked to different cancer-related processes. The microRNA miR-21 appears to function as an anti-apoptosis factor in glioblastomas. However, the functional target genes of miR-21 are largely unknown in glioblastomas. In this study, bioinformatics analysis was used to identify miR-21 target sites in various genes. Luciferase activity assay showed that a number of genes involved in apoptosis, PDCD4, MTAP, and SOX5, carry putative miR-21 binding sites. Expression of PDCD4 protein correlates inversely with expression of miR-21 in a number of human glioblastoma cell lines such as T98G, A172, U87, and U251. Inhibition of miR-21 increases endogenous levels of PDCD4 in cell line T98G and over-expression miR-21 inhibits PDCD4-dependent apoptosis. Together, these results indicate that miR-21 expression plays a key role in regulating cellular processes in glioblastomas and may serve as a target for effective therapies.