Vitamin D3 suppresses the cholesterol homeostasis pathway in patient-derived glioma cell lines.

Glioblastoma is one of the most common malignant brain tumors. Vitamin D, primarily its hormonally active form calcitriol, has been reported to have anti-cancer activity. In the present study, we used patient-derived glioma cell lines to examine the effect of vitamin D3 and calcitriol on glioblastoma. Surprisingly, vitamin D3 showed a more significant inhibitory effect than calcitriol on cell viability and proliferation. Vitamin D receptor (VDR) mediates most of the cellular effects of vitamin D, and thus we examined the expression level and function of VDR via gene silencing and gene knockout experiments. We observed that VDR does not affect the sensitivity of patient-derived glioma cell lines to vitamin D3, and the gene encoding VDR is not essential for growth of patient-derived glioma cell lines. RNA sequencing data analysis and sterolomics analysis revealed that vitamin D3 inhibits cholesterol synthesis and cholesterol homeostasis by inhibiting the expression level of 7-dehydrocholesterol reductase, which leads to the accumulation of 7-dehydrocholesterol and other sterol intermediates. In conclusion, our results suggest that vitamin D3, rather than calcitriol, inhibits growth of patient-derived glioma cell lines via inhibition of the cholesterol homeostasis pathway.

Primary Skull Base Chordomas: A Clinicopathological Analysis of 94 Patients.

OBJECTIVE: To investigate prognostic factors in patients with primary skull base chordoma (PSBC) to guide future therapeutic advances. METHODS: This retrospective cohort study of 94 PSBC patients was conducted in 2 institutions from January 2006 to December 2013. Independent predictors for progression-free survival (PFS) and overall survival were established with multivariate Cox regression analysis. RESULTS: Age (P = 0.006), extent of resection (P = 0.037), and radiotherapy (RT) (P = 0.027) were established as independent predictors for PFS in PSBC patients. Similarly, age (P = 0.002), extent of resection (P = 0.048), and RT (P = 0.015) were established as independent predictors for overall survival. Meta-analysis manifested that lower MIB-1 correlated with longer PFS in skull base chordoma patients (P < 0.001). RT doubled the 5-year PFS rate from 28.6 ± 12.1% to 61.6 ± 10.7% (P = 0.031) and increased the 5-year overall survival rate from 54.5 ± 13.8% to 84.2 ± 8.4% (P = 0.020) in the subtotal resection/partial resection and MIB-1 labeling index (STR/PR+MIB-1 LI) <2% subgroup. In contrast, in the STR/PR+MIB-1 LI ≥2% subgroup, the survival benefit of RT remained uncertain. Further analysis revealed no survival difference between different RT modalities in STR/PR PSBC patients. CONCLUSIONS: In PSBC patients, age, extent of resection, and adjuvant RT all are independent predictors for PFS. Lower MIB-1 LI is associated with longer PFS in PSBC patients. Adjuvant RT is necessary for PSBC patients who undergo STR/PR with MIB-1 LI <2%. Patients who undergo GTR or STR/PR with MIB-1 LI ≥2% seem nonresponsive to RT.

Increased DKC1 expression in glioma and its significance in tumor cell proliferation, migration and invasion.

The dyskeratosis congenita 1 (DKC1) gene is located on the X chromosome at Xq28. Dyskerin encoded by the DKC1 gene is associated with the formation of certain small RNAs and the telomerase activity. Inherited mutations in DKC1 inactivate the dyskerin and causes dyskeratosis congenital, which is characterized by skin defects, hematopoiesis failure, and increased susceptibility to cancer. DKC1 reportedly up-regulates in several human cancers, including renal cell carcinoma and prostate cancer. Dyskerin is deregulated in B-chronic lymphocytic leukemia and breast carcinomas, but its expression and function in glioma have hardly been investigated. Hence, we were prompted to collect tissue samples and implement cell experiments. Our study reveals that DKC1 expression is significantly increased in the pathological tissues of glioma compared with that in normal tissues. The increased staining of DKC1 is related to the World Health Organization stages of tumors. DKC1 knockdown also significantly inhibits glioma cell growth by altering the expression of cell cycle-relative molecules to arrest at the G1 phase. In the transwell chamber, DKC1 knockdown glioma cells exhibit low motility. Consistent with classic oncogenic pathways, N-cadherin, HIF-1α, and MMP2 expression levels are lower compared with those of the control group. Therefore, DKC1 up-regulation in gliomas is common and necessary for extensive tumor growth. The phenotype of glioma cell lines after DKC1 down-regulation suggests its use as a valuable clinical treatment strategy.

A pseudogene-signature in glioma predicts survival.

Pseudogene was recognized as a potential tumor suppressor or oncogene in varies of diseases, however its roles in glioma have not been investigated. Our study was to identify the pseudogene-signature that predicted glioma survival. Using a pseudogene-mining approach, we performed pseudogene expression profiling in 183 glioma samples from the Chinese Glioma Genome Atlas (CGGA) and set it as the training set. We found a six-pseudogene signature correlated with patients' clinical outcome via bioinformatics analyses (P ≤ 0.01), and validated it in the Repository of Molecular Brain Neoplasia Data (REMBRANDT) containing 350 cases. A formula calculating the risk score based on the six-pseudogene signature was introduced and the patients of CGGA set were classified into high-risk group and low-risk group with remarkably different survival (P < 0.001) based on their scores. The prognostic value of the signature was confirmed in the REMBRANDT set. Though the function of these pseudogenes is not clear, the identification of the prognostic pseudogenes indicated the potential roles of pseudogenes in glioma pathogenesis and they may have clinical implications in treating glioma.

MiR-1224-5p acts as a tumor suppressor by targeting CREB1 in malignant gliomas.

The dysregulation of miR-1224-5p has been reported in several human cancers. However, the expression and function of miR-1224-5p in glioma remains unknown. The aim of our study was to investigate the effect of miR-1224-5p on glioma cells and to determine its functional signaling mediators. Using 198 glioma samples within the Chinese Glioma Genome Atlas expression dataset, we demonstrated that miR-1224-5p expression is decreased in high-grade gliomas when compared with low-grade gliomas. Differential miR-1224-5p expression in 50 randomly selected samples was verified by in situ hybridization. The expression of miR-1224-5p was shown to positively correlate with overall survival in 82 glioblastoma patients. Exogenous expression of miR-1224-5p in glioma cells suppressed proliferation and invasion and promoted apoptosis. Target prediction algorithms identified a consensus miR-1224-5p recognition site in the 3'UTR of the cAMP response element-binding protein (CREB1) gene, and this sequence was shown to directly confer miR-1224-5p repression in luciferase reporter assays. Furthermore, exogenous miR-1224-5p expression was shown to down-regulate CREB1, as well as its downstream target genes matrix metalloproteinase-9 and B-cell lymphoma-2. Conversely, over-expression of CREB1 reversed the effect of miR-1224-5p on the proliferation, invasion, and apoptosis of glioma cells. These data indicate that miR-1224-5p may inhibit tumor-associated activity in malignant gliomas by targeting CREB1.

Fenofibrate induces G0/G1 phase arrest by modulating the PPARα/FoxO1/p27 kip pathway in human glioblastoma cells.

Fenofibrate, a fibric acid derivative, is known to possess lipid-lowering effects. Although fenofibrate-induced peroxisome proliferator-activated receptor alpha (PPARα) transcriptional activity has been reported to exhibit anticancer effects, the underlying mechanisms are poorly understood. In this study, we investigated the mechanisms behind the antiproliferative effects of fenofibrate in U87MG cells (human glioma cell line) using the WST-8 Cell Proliferation Assay Kit. Furthermore, we examined genome-wide gene expression profiles and molecular networks using the DAVID online software. Fenofibrate reduced the expression of 405 genes and increased the expression of 2280 genes. DAVID analysis suggested that fenofibrate significantly affected cell cycle progression and pathways involved in cancer, including the mTOR signaling pathway and insulin signaling pathway. Results of flow cytometry analysis indicated that fenofibrate induced cell cycle G0/G1 arrest in U87MG cells. Furthermore, we identified the FoxO1-p27(kip) signaling axis to be involved in fenofibrate-induced cell cycle arrest. Our findings suggest that in addition to its known lipid-lowering effects, fenofibrate may be used as an antitumor agent in glioma therapy.

RNA-seq of 272 gliomas revealed a novel, recurrent PTPRZ1-MET fusion transcript in secondary glioblastomas.

Studies of gene rearrangements and the consequent oncogenic fusion proteins have laid the foundation for targeted cancer therapy. To identify oncogenic fusions associated with glioma progression, we catalogued fusion transcripts by RNA-seq of 272 gliomas. Fusion transcripts were more frequently found in high-grade gliomas, in the classical subtype of gliomas, and in gliomas treated with radiation/temozolomide. Sixty-seven in-frame fusion transcripts were identified, including three recurrent fusion transcripts: FGFR3-TACC3, RNF213-SLC26A11, and PTPRZ1-MET (ZM). Interestingly, the ZM fusion was found only in grade III astrocytomas (1/13; 7.7%) or secondary GBMs (sGBMs, 3/20; 15.0%). In an independent cohort of sGBMs, the ZM fusion was found in three of 20 (15%) specimens. Genomic analysis revealed that the fusion arose from translocation events involving introns 3 or 8 of PTPRZ and intron 1 of MET. ZM fusion transcripts were found in GBMs irrespective of isocitrate dehydrogenase 1 (IDH1) mutation status. sGBMs harboring ZM fusion showed higher expression of genes required for PIK3CA signaling and lowered expression of genes that suppressed RB1 or TP53 function. Expression of the ZM fusion was mutually exclusive with EGFR overexpression in sGBMs. Exogenous expression of the ZM fusion in the U87MG glioblastoma line enhanced cell migration and invasion. Clinically, patients afflicted with ZM fusion harboring glioblastomas survived poorly relative to those afflicted with non-ZM-harboring sGBMs (P < 0.001). Our study profiles the shifting RNA landscape of gliomas during progression and reveled ZM as a novel, recurrent fusion transcript in sGBMs.

MiR-143 acts as a tumor suppressor by targeting N-RAS and enhances temozolomide-induced apoptosis in glioma.

Therapeutic applications of microRNAs (miRNAs) in RAS-driven glioma were valuable, but their specific roles and functions have yet to be fully elucidated. Here, we firstly report that miR-143 directly targets the neuroblastoma RAS viral oncogene homolog (N-RAS) and functions as a tumor-suppressor in glioma. Overexpression of miR-143 decreased the expression of N-RAS, inhibited PI3K/AKT, MAPK/ERK signaling, and attenuated the accumulation of p65 in nucleus of glioma cells. In human clinical specimens, miR-143 was downregulated where an adverse with N-RAS expression was observed. Furthermore, overexpression of miR-143 decreased glioma cell migration, invasion, tube formation and slowed tumor growth and angiogenesis in a manner associated with N-RAS downregulation in vitro and in vivo. Finally, miR-143 also sensitizes glioma cells to temozolomide (TMZ),the first-line drug for glioma treatment. Taken together, for the first time, our results demonstrate that miR-143 plays a significant role in inactivating the RAS signaling pathway through the inhibition of N-RAS, which may provide a novel therapeutic strategy for treatment of glioma and other RAS-driven cancers.

Inhibition of activated Ras suppresses multiple oncogenic Hub genes in human epithelial tumors.

Cancer cells may involve diverse mutations, but they often rely on continued expression of a single oncoprotein for survival, as a response to targeting this protein. Generally, Ras is overexpressed in human epithelial tumors and cancellation of activated Ras inhibits carcinoma cell proliferation and differentiation ability, and induces apoptotosis of tumor cells. However, the mechanisms of inhibition of activated Ras that suppress the malignancy activity of human epithelial tumors remain to be illuminated. We utilized text-mining of MEDLINE abstracts with natural language processing to establish the Ras biologic association network, and identified several interactions of this network with the Ras pathway. Our investigation not only examined the expression of Ras and Hub genes (PIK3CA, MDM2, CCND1, EGFR, JUN, MYC, VEGFA, ERK1 and ERK2) but also confirmed inhibition of activated Ras reduced expression of multiple oncogene in vitro studies. Our studies provide strong support for the conclusion that cancellation of activated Ras specifically regulates defective Ras pathways in human tumor cells.

Long noncoding RNA profiles reveal three molecular subtypes in glioma.

BACKGROUND: Gliomas are the most lethal type of primary brain tumor in adult. Long noncoding RNAs (lncRNAs), which are involved in the progression of various cancers, may offer a potential gene therapy target in glioma. METHODS AND FINDINGS: We first classified gliomas into three molecular subtypes (namely LncR1, LncR2 and LncR3) in Rembrandt dataset using consensus clustering. Survival analysis indicated that LncR3 had the best prognosis, while the LncR1 subtype showed the poorest overall survival rate. The results were further validated in an independent glioma dataset GSE16011. Additionally, we collected and merged data of the two databases (Rembrandt and GSE16011 dataset) and analyzed prognosis of each subtype in WHO II, III and IV gliomas. The similar results were obtained. Gene Set Variation Analysis (GSVA) demonstrated that LncR1 subtype enriched cultured astroglia's gene signature, while LncR2 subtype was characterized by neuronal gene signature. Oligodendrocytic was rich in LncR3. In addition, IDH1 mutation and 1p/19q LOH were found rich with LncR3, and EGFR amplification showed high percentage in LncR1 in GSE16011 dataset. CONCLUSIONS: We report a novel molecular classification of glioma based on lncRNA expression profiles and believe that it would provide a potential platform for future studies on gene treatment for glioma and lead to more individualized therapies to improve survival rates.

HOTAIR, a cell cycle-associated long noncoding RNA and a strong predictor of survival, is preferentially expressed in classical and mesenchymal glioma.

BACKGROUND: Long noncoding RNA Hox transcript antisense intergenic RNA (HOTAIR) has been characterized as a negative prognostic factor in breast and colon cancer patients. The clinical significance and function of HOTAIR in glioma remains unclear. METHODS: We analyzed the clinical significance of HOTAIR in 3 different glioma cohorts with gene expression data, including correlation with tumor grade, prognosis, and molecular subtype. The function of HOTAIR in glioma was explored by performing gene set enrichment analysis and in vitro and in vivo experiments. RESULTS: HOTAIR expression was closely associated with glioma grade and poor prognosis. Multivariate Cox regression analysis revealed that HOTAIR was an independent prognostic factor in glioblastoma multiforme patients. HOTAIR expression correlated with glioma molecular subtype, including those of The Cancer Genome Atlas. HOTAIR was preferentially expressed in the classical and mesenchymal subtypes compared with the neural and proneural subtypes. A gene set enrichment analysis designed to show gene set differences between patients with high and low HOTAIR expression indicated that HOTAIR expression was associated with gene sets involved in cell cycle progression. HOTAIR reduction induced colony formation suppression, cell cycle G0/G1 arrest, and orthotopic tumor growth inhibition. CONCLUSION: Our data establish that HOTAIR is an important long noncoding RNA that primarily serves as a prognostic factor for glioma patient survival, as well as a biomarker for identifying glioma molecular subtypes, a critical regulator of cell cycle progression.

Overexpressed let-7a inhibits glioma cell malignancy by directly targeting K-ras, independently of PTEN.

BACKGROUND: Altered expression of micro(mi)RNAs has been shown to be associated with tumorigenesis and tumor progression. The expression of phosphatase and tensin homolog (PTEN) plays an important role in glioma and is regarded as a prognostic marker of glioma patients. The goal of this study was to investigate the function of lethal (let)-7a miRNA in glioma cell lines with different PTEN phenotypes. METHODS: One hundred ninety-eight glioma tissues were used to profile miRNA expression. RESULTS: Let-7a was shown to have lower expression in high-grade glioma than in low-grade glioma. Low expression of let-7a was correlated with poor prognosis of primary glioblastoma patients. We demonstrated that K-ras was a functional target for let-7a to induce cell cycle arrest, apoptosis, and inhibition of cell migration and invasion in vitro. Our further results showed no difference in malignancy inhibition induced by let-7a in 4 glioma cells, including U87 (PTEN null), U251 (PTEN mutant), LN229 (PTEN wild type), and LN229 (PTEN small interfering RNA). The phosphatidylinositol-3 kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase pathways were inhibited by let-7a, and the inhibition effects had no difference in 4 glioma cells. We demonstrated that let-7a could induce suppression of glioma in vivo by generating a glioma xenograft model. CONCLUSION: Our results indicated that let-7a suppresses its target transcript K-ras and inhibits glioma malignancy independent of PTEN expression.

BCL2A1 is a potential biomarker for postoperative seizure control in patients with low-grade gliomas.

AIMS: To identify molecular genetic factors that influence preoperative seizure occurrence and postoperative seizure control in patients with low-grade gliomas (LGGs). METHODS: Fifty-four WHO grade II astrocytomas were used for microarray analysis under strict inclusion criteria. The primary endpoint was seizure control at 12 months after surgery. Biological processes were investigated by gene ontology (GO) analysis. Quantitative RT-PCR and immunohistochemistry were used to validate key genes. RESULTS: Differentially expressed genes correlated with seizure occurrence failed to significantly distinguish patients with and without a history of seizures. With respect to postoperative seizure control, a transcript profile of 92 genes was identified, which successfully separated patients with good and poor seizure prognosis. GO analysis revealed that the most striking overrepresentation of genes was found in a category of anti-apoptotic genes and their regulation. Increased expression was also observed for genes involved in immune and inflammatory responses. BCL2A1 was proven to be a novel marker associated with seizure prognosis. CONCLUSION: Increased anti-apoptotic activity of tumor cells appears to contribute to seizure recurrence after surgery in patients with LGGs. These findings provide insights that may lead to the development of effective treatment strategies for prolonging the survival of patients with LGG in the future.

Vestibular schwannoma surgical treatment.

Neurosurgical intervention remains the main step in the effective management of vestibular schwannomas. Extensive studies on vestibular schwannoma treatment have placed emphasis on preserving quality of life and neurological functions, particularly of the facial and vestibulocochlear nerves. Facial nerve preservation and hearing preservation have been achieved by significant advances in skull base microsurgical techniques and intraoperative neuromonitoring. Diffusion tensor imaging is a powerful and accurate method for preoperatively identifying the facial nerve in relation to vestibular schwannomas. Endoscopy offers excellent illumination of the anatomical structures and provides panoramic vision inside the surgical area. In this report, we focused on facial nerve and vestibulocochlear nerve preservation and analyzed the major techniques used for identifying the nerve-tumor relationship.

MiRNA-181b suppresses IGF-1R and functions as a tumor suppressor gene in gliomas.

MicroRNAs (miRNAs) are single-stranded, 18- to 23-nt RNA molecules that function as regulators of gene expression. Previous studies have shown that microRNAs play important roles in human cancers, including gliomas. Here, we found that expression levels of miR-181b were decreased in gliomas, and we identified IGF-1R as a novel direct target of miR-181b. MiR-181b overexpression inhibited cell proliferation, migration, invasion, and tumorigenesis by targeting IGF-1R and its downstream signaling pathways, PI3K/AKT and MAPK/ERK1/2. Overexpression of IGF-1R rescued the inhibitory effects of miR-181b. In clinical specimens, IGF-1R was overexpressed, and its protein levels were inversely correlated with miR-181b expression. Taken together, our results indicate that miR-181b functions in gliomas to suppress growth by targeting the IGF-1R oncogene and that miR-181b may serve as a novel therapeutic target for gliomas.

Unique genome-wide map of TCF4 and STAT3 targets using ChIP-seq reveals their association with new molecular subtypes of glioblastoma.

BACKGROUND: Aberrant activation of beta-catenin/TCF4 and STAT3 signaling in glioblastoma multiforme (GBM) has been reported. However, the molecular mechanisms related to this process are still poorly understood. METHODS: Genome-wide screening of the binding characteristics of the transcription factors TCF4 and STAT3 in GBM cells was performed by chromatin immunoprecipitation sequencing (ChIP-seq) assay. Hierarchical clustering was used to analyze the association of TCF4 and STAT3 coregulated genes with The Cancer Genome Atlas (TCGA) GBM subtypes (classical, mesenchymal, neural, and proneural). New molecular classification of GBM was proposed and validated in Western and Asian populations. RESULTS: We identified 1250 overlapping putative target genes that were coregulated by TCF4 and STAT3. Further, the coregulated genes had the potential to guide TCGA GBM subtypes. Finally, we proposed a new molecular classification of GBM into 2 subtypes (proneural-like and mesenchymal-like) and showed that the new classification could be applied to both Western and Asian populations. In addition, the GBM response to temozolomide therapy differed depending on its subtype; mesenchymal-like GBM benefited, while there was no benefit for proneural-like GBM. CONCLUSIONS: This is the first comprehensive study to combine a ChIP-seq assay of TCF4 and STAT3 and data mining of patient cohorts to derive molecular subtypes of GBM.

MiR-21 modulates hTERT through a STAT3-dependent manner on glioblastoma cell growth.

BACKGROUND AND PURPOSE: As an important oncogenic miRNA, miR-21 has been reported to play crucial roles in glioblastoma (GBM) carcinogenesis. However, the precise biological function and molecular mechanism of miR-21 in GBM remain elusive. This study is designed to explore the mechanism of miR-21 involved in the control of GBM cell growth. METHODS AND RESULTS: MTT assay, cell cycle analysis, and apoptosis analysis showed that reduction of miR-21 inhibited cell growth in U87 and LN229 GBM cells. Further, reduction of miR-21 decreased the expression of human telomerase reverse transcriptase (hTERT) and repressed STAT3 expression and STAT3 phosphorylation. STAT3 inhibition led to a remarkable depletion of hTERT at both mRNA and protein levels by binding to the hTERT gene promoter by performing luciferase reporter assay and chromatin Immunoprecipitation PCR. Finally, knockdown of miR-21 considerably inhibited tumor growth and diminished the expression of STAT3 and hTERT in xenograft model. CONCLUSION: Our findings indicate that miR-21 regulates hTERT expression mediated by STAT3, therefore controlling GBM cell growth.

Association between the Ku70-1310C/G promoter polymorphism and cancer risk: a meta-analysis.

Ku70 plays an important role in DNA double-strand break repair. Studies revealing conflicting results on the role of the Ku70-1310C/G promoter polymorphism on cancer risk led us to perform a meta-analysis to investigate this relationship. Ten case-control studies with 2566 cases and 3058 controls were identified. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the strength of associations. The overall results suggested no association between the Ku70-1310C/G promoter polymorphism and total cancer risk. However, on stratified analysis, significantly increased risks were observed among the Asian population (GG vs. CC: OR=1.50, 95%CI=1.10-2.06; GG vs. CC/CG: OR=1.47, 95%CI=1.07-2.01) and population-based case- control studies (GG vs. CC: OR=1.57, 95%CI=1.12-2.22; CG vs. CC: OR=1.35, 95%CI=1.11-1.64; CG/GG vs. CC: OR=1.37, 95%CI=1.14-1.65). Additionally, variant genotypes were associated with a significantly increased breast cancer risk (GG vs. CC: OR=1.80, 95%CI=1.26-2.56; GG vs. CC/CG: OR=1.40, 95%CI=1.01-1.95).