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Glioma is the most common primary malignant tumor in the central nervous system, and directly affects the quality of life and cognitive function of patients. Ferroptosis, is a new form of regulated cell death characterized by iron-dependent lipid peroxidation. Ferroptosis is mainly due to redox imbalance and involves multiple intracellular biology processes, such as iron metabolism, lipid metabolism, and antioxidants synthesis. Induction of ferroptosis could be a new target for glioma treatment, and ferroptosis-related processes are associated with chemoresistance and radioresistance in glioma. In the present review, we provide the characteristics, key regulators and pathways of ferroptosis and the crosstalk between ferroptosis and other programmed cell death in glioma, we also proposed the application and prospect of ferroptosis in the treatment of glioma.

BACKGROUND Dysregulation of immune checkpoint molecules leads to immune evasion in human tumors but has become a viable target for tumor therapy. Here, we examined expression of LIGHT (TNFSF14), an immune checkpoint molecule, in human glioblastoma (GBM) to assess its potential as a molecular target for treatment. METHODS Molecular and clinical data from publicly available genomic databases containing WHO grade II-IV human glioma cases were analyzed. Immunohistochemistry was applied to assess LIGHT protein levels in primary tumor sections. Statistical analysis was performed using Matlab and R language. FINDINGS LIGHT was found to be elevated in aggressive gliomas, particularly in the mesenchymal molecular subtype and isocitrate dehydrogenase (IDH) wild-type GBM. LIGHT exhibited potential as a prognostic marker based on Cox regression and nomogram models. LIGHT displayed intra-tumor heterogeneity and was more highly expressed in peri-necrotic and microvascular regions. Gene ontology and pathway analysis revealed enrichment of LIGHT in multiple immune regulatory processes. Finally, in cell lineage analysis, LIGHT was found to be tightly associated with several infiltrating immune and stromal cell types which localized to the tumor microenvironment. INTERPRETATION Our data highlights the importance of LIGHT in the development of GBM and as a potential molecular target in combination with current immune checkpoint blockades for treatment of GBM.

BACKGROUND Glioblastoma (GBM) is the most malignant primary tumor of the central nervous system, while the pathogenesis remains unclear. Protein lysine methyltransferase SETD8, which is responsible for the modification of histone H4K20me1, has been shown to play an important role in cellular transcriptional regulation and the development of a variety of tumors, yet its role in the malignant progression of GBM has not been elucidated. MATERIAL AND METHODS In the present study, we used primary GBM cell lines, intracranial xenograft model, transcriptome sequencing together with ChIP-sequencing, aiming to elucidate the molecular mechanism of SETD8-mediated H4K20me1 transcriptional regulation in promoting GBM progression. Furthermore, we evaluated the potential therapeutic significance in GBM using SETD8 small molecule inhibitor, UNC0379. RESULTS We found that SETD8 is aberrantly expressed in GBM tissues, accompanied by the dysregulation of H4K20me1 modification, which is associated with tumor pathology and prognosis. Using SETD8 inhibitor UNC0379 or knockdown of SETD8 significantly inhibited GBM cell proliferation in vitro and in vivo, and downregulated H4K20me1 modification level as well as transcriptome expression. CONCLUSION In summary, our work provide a novel insight into the role of SETD8/H4K20me1 axis. SETD8 overexpression mediated aberrant H4K20me1 modification act as a novel "transcriptional switch" in the malignant progression of glioma.

BACKGROUND: Gliomas are the most common primary malignant brain tumor. Diffuse low-grade and intermediate-grade gliomas, which together compose the lower-grade gliomas (LGGs; World Health Organization [WHO] grades II and III), present a therapeutic challenge to physicians due to the heterogeneity of their clinical behavior. Nomograms are useful tools for individualized estimation of survival. This study aimed to develop and independently validate a survival nomogram for patients with newly diagnosed LGG. METHODS: Data were obtained for newly diagnosed LGG patients from The Cancer Genome Atlas (TCGA) and the Ohio Brain Tumor Study (OBTS) with the following variables: tumor grade (II or III), age at diagnosis, sex, Karnofsky performance status (KPS), and molecular subtype (IDH mutant with 1p/19q codeletion [IDHmut-codel], IDH mutant without 1p/19q codeletion, and IDH wild-type). Survival was assessed using Cox proportional hazards regression, random survival forests, and recursive partitioning analysis, with adjustment for known prognostic factors. The models were developed using TCGA data and independently validated using the OBTS data. Models were internally validated using 10-fold cross-validation and externally validated with calibration curves. RESULTS: A final nomogram was validated for newly diagnosed LGG. Factors that increased the probability of survival included grade II tumor, younger age at diagnosis, having a high KPS, and the IDHmut-codel molecular subtype. CONCLUSIONS: A nomogram that calculates individualized survival probabilities for patients with newly diagnosed LGG could be useful to health care providers for counseling patients regarding treatment decisions and optimizing therapeutic approaches. Free online software for implementing this nomogram is provided: https://hgittleman.shinyapps.io/LGG_Nomogram_H_Gittleman/. KEY POINTS: 1. A survival nomogram for lower-grade glioma patients has been developed and externally validated. 2. Free online software for implementing this nomogram is provided allowing for ease of use by practicing health care providers.

Galangin (GG), a flavonoid, elicits a potent antitumor activity in diverse cancers. Here, we evaluated the efficacy of GG in the treatment of human glioblastoma multiforme (GBM) and investigated the molecular basis for its inhibitory effects in the disease. GG inhibited viability and proliferation of GBM cells (U251, U87MG, and A172) in a dose-dependent manner (IC50 = 221.8, 262.5, 273.9 μM, respectively; P < 0.001; EdU, ~40% decrease at 150 μM, P < 0.001), and the number of colonies formed was significantly reduced (at 50 μM, P < 0.001). However, normal human astrocytes were more resistant to its cytotoxic effects (IC50 >450 μM). Annexin-V/PI staining was increased indicating that GG induced apoptosis in GBM cells (26.67 and 30.42%, U87MG and U251, respectively) and associated proteins including BAX and cleaved PARP-1 were increased (~3×). Cells also underwent pyroptosis as determined under phase-contrast microscopy. Knockdown of gasdermin E (GSDME), a protein involved in pyroptosis, alleviated pyroptosis induced by GG through aggravating nuclear DNA damage in GBM cells. Meanwhile, fluorescent GFP-RFP-MAP1LC3B puncta associated with autophagy increased under GG treatment, and transmission electron microscopy confirmed the formation of autophagic vesicles. Inhibition of autophagy enhanced GG-induced apoptosis and pyroptosis in GBM cells. Finally, in an orthotopic xenograft model in nude mice derived from U87MG cells, treatment with GG in combination with an inhibitor of autophagy, chloroquine, suppressed tumor growth, and enhanced survival compared to GG monotherapy (P < 0.05). Our results demonstrated that GG simultaneously induces apoptosis, pytoptosis, and protective autophagy in GBM cells, indicating that combination treatment of GG with autophagy inhibitors may be an effective therapeutic strategy for GBM. publishedVersion

The nomogram represents a statistical model that incorporates multiple risk factors to estimate individualized survival probabilities. In this study, we developed a nomogram which provides an important tool for individulized survival predicition for newly diagnosed low-grade gliomas (LGG). A total number of 582 newly diagnosed LGG patients were included; the median age was 39.93 years and 42% were female. Cox regression analysis showed that younger age at diagnosis, WHO grade II vs. III, the IDHmut-codel vs. the IDHwt, and the IDHmut-non-codel vs. the IDHwt were significantly associated with better prognosis. The adjuvant treatment following surgery showed a trend towards improved survival. Subsequently, the nomogram to estimate 60-, 90-, and 120-month survival probabilities was established. Our data showed that the age at diagnosis was the largest contributor to patient survival, followed by molecular subtype, WHO grade, treatment and gender. The calibration plot showed that the observed and the nomogram predicted OS curves were well-aligned. In addition, we also validated our nomogram for LGG patients who received postsurgical adjuvant therapy through cross-validation and the calibration plot. Finally, we developed a free online tool for this nomogram (softwarewebsite:https://rrlnnomogram.shinyapps.io/LGG_Nom_Asian/). Overall, this model should be a useful tool for counseling patients in clinical practice including treatment decisions, follow-up, and prognosis.

The Ras-related GTP-binding protein (RAB) family plays an important role in regulating signal transduction and cellular processes including vesicle transport, cytoskeleton formation and membrane trafficking. More recently, several RAB members have been reported to promote tumorigenesis in many types of cancers. However, the clinical significance and potential function of RAB43 in gliomas remain unclear. Herein, we found that RAB43 was upregulated and positively correlated with the grade of progression in glioma patients by in silico analysis and immunohistochemistry (IHC). Patients with high RAB43 displayed worse clinical outcomes in comparison to those with low RAB43. RAB43 was also highly expressed in mesenchymal and G3 subtypes, and isocitrate dehydrogenase 1 (IDH1) wild-type gliomas. Moreover, transcriptomic analyses via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways revealed that RAB43‑related gene sets were mainly involved in the regulation of cell adhesion and cell migration processes. Further investigation indicated that RAB43 downregulation significantly suppressed the migratory and invasive ability of glioma cells, as well as decreased the expression of epithelial-mesenchymal transition (EMT) markers (N-cadherin, vimentin and Snail). In conclusion, a high level of RAB43 was significantly associated with the malignant phenotypes of gliomas, which suggests that RAB43 may serve as a novel biomarker and a potential therapeutic target for gliomas.

BACKGROUND: Long noncoding RNAs (lncRNAs) are essential transcripts with critical roles in tumor initiation and malignant progression. In the present study, through analysis of whole-transcriptome profiles, we showed that MIR22 host gene (MIR22HG), the primary microRNA of miR-22, ranked among the most overexpressed lncRNAs in glioblastoma (GBM). Herein, our study aims to elucidate the biological functions of MIR22HG and therapeutic potency of MIR22HG targeted inhibition in GBM. METHODS: We characterized the expression pattern of MIR22HG/miR-22 by bioinformatics analysis and by in situ hybridization (ISH) in GBMs. We further determined whether genetic targeting of MIR22HG affected oncogenic functions of primary GBM cells in vitro and in vivo. A co-culture model where brain organoids are co-cultured with GBM spheroids were utilized for the invasion assessment. Finally, based on the three-dimensional structure of the pre-miR-22, we performed virtual screening strategy to disclose a novel inhibitor of the MIR22HG/miR-22 axis. RESULTS: We showed that MIR22HG/miR-22 was highly expressed in GBM, especially in isocitrate dehydrogenase 1/2 (IDH1/2) wild-type tumors. Specially, MIR22HG/miR-22 was preferentially expressed in glioblastoma stem cells (GSCs) compared to normal neural stem cell (NSCs). Analysis of intra-tumoral transcriptional heterogeneity showed that MIR22HG was higher in the infiltrating region compared to the tumor center. In functional assays, silencing MIR22HG attenuated aggressive phenotypes in GBM cells, including invasion, self-renewal and in vivo tumor growth via suppressing the production of miR-22-3p and miR-22-5p. Using a luciferase reporter assay, we further confirmed that two inhibitors of the Wnt/β-catenin pathway, SFRP2 and PCDH15, were direct targets of miR-22-3p and miR-22-5p. Finally, we identified a specific inhibitor termed AC1L6JTK, which blocks the ability of Dicer to process pre-miR-22 to mature miR-22, thus exerting therapeutic efficacy on GBMs. CONCLUSIONS: Our findings show that the MIR22HG/miR-22 axis is a novel biomarker as well as a therapeutic target in GBM.

BACKGROUND: Tumor cells display higher cholesterol levels compared to normal cells and emerging evidence indicates a pivotal role of cholesterol content in tumor cell proliferation and growth mediated by a dysregulation of key regulators involved in cholesterol homeostasis has been implicated in various cancers. In this study, we assessed the dependency of glioblastoma (GBM) cells to extracellular cholesterol levels for growth. Through analysis of whole-transcriptome databases, we identified the cholesterol homeostasis-related genes whose expression are dysregulated in gliomas. One of the most dramatically down-regulated genes identified encodes cholesterol 24-hydroxylase (CYP46A1), a brain-specific enzyme responsible for elimination of cholesterol via conversion of cholesterol to 24(S)-hydroxycholesterol (24OHC). MATERIAL AND METHODS: Molecular and clinical data was obtained from The Cancer Genome Atlas (TCGA) database. Immunohistochemistry was applied to assess the protein level of CYP46A1 in tissue microarrays. Functionally, CYP46A1 overexpression was determined using lentiviral constructs in LN229 and a primary glioblastoma cell line (GBM#P3). Cells were characterized in vitro and implanted in vivo to generate orthotopic xenografts in order to assess their molecular status, cell survival, and self-renewal by western blotting, flow cytometry, and extreme limiting dilution assay (ELDA), respectively. RESULTS: Down-regulation of CYP46A1 was found in malignant glioma samples compared to normal brain tissue. Importantly, reduced CYP46A1 expression was associated with aggressive clinicopathological features and poor prognosis of GBM patients. In functional assays, restoration of CYP46A1 suppressed cell proliferation and in vivo tumour growth by increasing 24OHC levels. Consistent, treatment of GBM cells with 24OHC inhibited tumour growth and induced apoptosis in a dose dependent manner. Mechanistically, 24OHC incubation reduced cellular cholesterol levels by suppressing the activation of the sterol regulatory-element binding protein 2 (SREBP2), thus downregulating downstream targets including low-density lipoprotein receptor (LDLR). Intriguingly, efavirenz (EFV), a widespread used anti-HIV medication that effectively penetrates the blood-brain barrier, has recently been reported as a potent activator of the CYP46A1 enzyme. The therapeutic potential of EFV is currently being assessed in vivo and the data will be presented. CONCLUSION: Collectively, our findings demonstrate that CYP46A1 is a critical cellular cholesterol regulator in GBM and that the CYP46A1/24OHC axis is a potential therapeutic target in GBM.

BACKGROUND: We have previously developed a brain organoid system based on the development of fetal rat brain cells taken from fetuses at the 18th day of gestation. The purpose of the present work was to use the latest technologies to assess in detail the differentiation of various neural cell lineages into mature brain organoids and also to assess their cellular organization into mature brain structures. We also explored in detail glioma cell invasion parameters such as speed of invasion, the clonal composition of invasive cells as their transcriptional profiles. MATERIAL AND METHODS: Western blot, immunohistochemistry, and immunofluorescence were used to detect the expression and distribution of mature neurons, astrocytes, oligodendrocytes, and microglia in the brain organoids at different stages of development. Transcriptomic analysis was also assessed by RNA-seq. To determine the invasive potential of the tumor cells into the organoids, we barcoded various tumor cell clones in order to establish a “rainbow” tumors where different clones were given a different color. By advanced imaging we tracked in real time the invasion of the barcoded cells. Moreover, tumor cells from the invasive areas as well as from the tumor core were analyzed by RNA-seq. RESULTS: Western blots showed that markers of mature neurons, astrocytes, oligodendrocytes and microglia were highly expressed at 14-21th day of brain organoid development, which was also reflected by RNA-seq. Having described in detail the mature organoid structure, we explored the invasive capacity and speed of invasion of the barcoded cells. Our results show that the average speed of glioma cell invasion is in the range of ~ 20 μm/h. We also show that that different glioma stem cell lines show different invasive capacities based on the tumor of origin. Genomic analysis of invasive cells revealed clear invasive signatures. CONCLUSION: We found that brain organoids display a highly cellular and structural organization. For the first time the speed of invasion was determined as well as their clonal composition. By a molecular identification of invasive and non-invasive cells new therapeutic strategies can be designed targeting the invasive compartment in GBMs.

BACKGROUND Glioblastoma (GBM) cell infiltration into the surrounding normal brain tissue where the blood brain barrier is intact, represents a major problem for clinical management and therapy. There is a vital need to understand the molecular mechanism that drives tumor cell invasion into the surrounding brain. We have previously developed a 3D coculture model where mature brain organoids are confronted with patient-derived glioblastoma stem-like cells (GSCs). In such a coculture system, single cell invasion into the normal brain tissue can be studied in detail. Here, we first describe in detail, by RNA-seq and proteomics, the differentiation of various neural cell lineages into mature brain organoids as well as their cellular organization. By real-time confocal microscopy and imaging analyses we also determine the speed of tumor cell invasion into the brain. Finally, we used this coculture system to delineate in detail the cellular heterogeneity within the invasive compartment and their gene expression. MATERIAL AND METHODS Immunohistochemistry and immunofluorescence were used to determine the expression and distribution of mature neurons, astrocytes, oligodendrocytes, and microglia within the brain organoids. Proteomics and RNA-seq were used to determine brain development ex-vivo. To assess the clonal composition of the GBM-invasive compartment, we used cellular (RGB) barcoding technology. By advanced imaging, we tracked in real time the invasion of barcoded cells into the brain organoids. Finally, we isolated invasive cells and non-invasive cells from our coculture system and used single cell sequencing to analyze their gene expression profiles and molecular phenotypes. RESULTS Immunohistochemistry and immunofluorescence showed that brain organoids, after 21 days of differentiation, display a highly cellular and structural organization. RNA-seq and proteomics, performed at different time points of organoid differentiation, revealed that the brain organoids develop into mature brain structures after 21 days as verified by a comparative analysis to normal rat brain development in vivo. Imaging analyses showed that multiple clones within the GBMs have the capacity to invade into the brain tissue with an average speed of ~ 20 μm/h. RNA-sec analysis of the invasive compartment revealed a strong up-regulation of genes and pathways associated with anaerobic respiration (glycolysis). CONCLUSION We describe a highly standardized brain organoid coculture system that can be used to delineate GBM invasion ex-vivo. We demonstrate that this platform can be used to unravel the mechanisms that drive GBM invasion into the normal brain.

Background Golgi Membrane Protein 1 (GOLM1), a protein involved in the trafficking of proteins through the Golgi apparatus, has been shown to be oncogenic in a variety of human cancers. Here, we examined the role of GOLM1 in the development of human glioma. Methods qRT-PCR, immunohistochemistry, and western blot analysis were performed to evaluate GOLM1 levels in cell lines and a cohort of primary human glioma and non-neoplastic brain tissue samples. Glioma cell lines were modified with lentiviral constructs expressing short hairpin RNAs targeting GOLM1 or overexpressing the protein to assess function in proliferation, viability, and migration and invasion in vitro using EdU, CCK8, clone-forming, Transwell assays, 3D tumor spheroid invasion assay and in vivo in orthotopic implantations. Protein lysates were used to screen a membrane-based antibody array to identify kinases mediated by GOLM1. Specific inhibitors of PDGFRα (AG1296) and AKT (MK-2206) were used to examine the regulation of PDGFA/PDGFRα on GOLM1 and the underlying pathway respectively. Results qRT-PCR, immunohistochemistry and western blot analysis revealed GOLM1 expression to be elevated in glioma tissues and cell lines. Silencing of GOLM1 attenuated proliferation, migration, and invasion of U251, A172 and P3#GBM (primary glioma) cells, while overexpression of GOLM1 enhanced malignant behavior of U87MG cells. We further demonstrated that activation of AKT is the driving force of GOLM1-promoted glioma progression. The last finding of this research belongs to the regulation of PDGFA/PDGFRα on GOLM1, while GOLM1 was also a key element of PDGFA/PDGFRα-mediated activation of AKT, as well as the progression of glioma cells. Conclusions PDGFA/PDGFRα-regulated GOLM1 promotes glioma progression possibly through activation of a key signaling kinase, AKT. GOLM1 interference may therefore provide a novel therapeutic target and improve the efficacy of glioma treatment, particularly in the case of the proneural molecular subtype of human glioma. Electronic supplementary material The online version of this article (10.1186/s13046-017-0665-3) contains supplementary material, which is available to authorized users.

Background Transgelin-2 (TAGLN2) is a member of the calponin family of actin-bundling proteins that is involved in the regulation of cell morphology, motility, and cell transformation. Here, the clinical significance and potential function of TAGLN2 in malignant gliomas were investigated. Methods Molecular and clinical data was obtained from The Cancer Genome Atlas (TCGA) database. Gene ontology and pathway analysis was used to predict potential functions of TAGLN2. RNA knockdown was performed using siRNA or lentiviral contructs in U87MG and U251 glioma cell lines. Cells were characterized in vitro or implanted in vivo to generate orthotopic xenografts in order to assess molecular status, cell proliferation/survival, and invasion by Western blotting, flow cytometry, and 3D tumor spheroid invasion assay, respectively. Results Increased TAGLN2 expression was associated with increasing tumor grade (P

Background: Resistance to adjuvant radiotherapy is a major cause of treatment failure in patients with glioblastoma (GBM). Autophagy inhibitors have been shown to enhance the efficacy of radiotherapy for certain solid tumors. However, current inhibitors do not penetrate the blood-brain-barrier (BBB). Here, we assessed the radiosensitivity effects of the antipsychotic drug trifluoperazine (TFP) on GBM in vitro and in vivo. Methods: U251 and U87 GBM cell lines as well as GBM cells from a primary human biopsy (P3), were used in vitro and in vivo to evaluate the efficacy of TFP treatment. Viability and cytotoxicity was evaluated by CCK-8 and clonogenic formation assays. Molecular studies using immunohistochemistry, western blots, immunofluorescence and qPCR were used to gain mechanistic insight into the biological activity of TFP. Preclinical therapeutic efficacy was evaluated in orthotopic xenograft mouse models. Results: IC50 values of U251, U87 and P3 cells treated with TFP were 16, 15 and 15.5 μM, respectively. TFP increased the expression of LC3B-II and p62, indicating a potential disruption of autophagy flux. These results were further substantiated by a decreased Lysotracker Red uptake, indicating impaired acidification of the lysosomes. We show that TFP and radiation had an additive effect when combined. This effect was in part due to impaired TFP-induced homologous recombination. Mechanistically we show that down-regulation of cathepsin L might explain the radiosensitivity effect of TFP. Finally, combining TFP and radiation resulted in a significant antitumor effect in orthotopic GBM xenograft models. Conclusions: This study provides a strong rationale for further clinical studies exploring the combination therapy of TFP and radiation to treat GBM patients. publishedVersion

Up to now, survivin has been recommended as a prognostic and diagnostic indicator in glioma patients. How- ever, there are still many controversies. Here, a meta-analysis was conducted to draw a more definitive conclusion on the correlation of survivin with overall survival (OS), age,gender, and WHO grade. Eligible studies were available through careful assessment, and then pooled hazard ratios (HRs) or odds ratios (ORs) with 95 % confidence intervals (95 % CIs) were estimated. Funnel plots were introduced to evaluate the publication bias. Additionally, heterogeneity and sensitivity were also evaluated. In the present meta-analysis, 15 eligible studies with a total of 1,089 patients were incorporated. Survivin expression in gliomas correlated with 2-year OS (n=8; HR 0.17, 95 % CI 0.11-0.26) and 5-year OS (n=7; HR 0.12, 95 % CI 0.07-0.22) in patients. In addition, a fixed-effect model revealed a significant asso- ciation between survivin and age (male/+; OR 2.10, 95 % CI 1.44-3.05) and survivin and WHO grade (I+II/+; OR 0.27, 95% CI 0.19-0.38).Noheterogeneitywasobservedacrossall studies. According to Begg' sa nd Egger's test and funnel plot, no publication bias was reported. Taken together, our meta- analysis suggests that survivin expression is associated with poor survival, older age, and higher WHO grade and could be suggested as a useful prognostic and diagnostic biomarker, or an effective therapy target.

BACKGROUND Dysregulated cholesterol metabolism is a hallmark of many cancers, including glioblastoma (GBM), but its role in disease progression is not well understood. Here, we identified cholesterol 24-hydroxylase (CYP46A1), a brain-specific enzyme responsible for elimination of cholesterol through conversion of cholesterol to 24(S)-hydroxycholesterol (24OHC), as one of the most dramatically dysregulated cholesterol metabolism genes in GBM. MATERIAL AND METHODS Molecular and clinical data was obtained from publicly genomic databases. Immunohistochemistry was applied to assess protein levels of CYP46A1 in primary GBM samples. Lentiviral constructs expressing CYP46A1 were transduced into LN229, LN18 and primary GBM GSCs for functional assays carried out in vitro and in vivo in an orthotopic xenograft model. RNA-seq was performed to identify downstream targets of 24OHC. RESULTS CYP46A1 was significantly decreased in GBM samples compared to normal brain tissue. Reduced CYP46A1 expression was associated with increasing tumour grade and poor prognosis in GBM patients. Ectopic expression of CYP46A1 suppressed cell proliferation and in vivo tumour growth by increasing 24OHC levels. Treatment of GBM cells with 24OHC suppressed tumour growth through regulation of LXR and SREBP signalling. Efavirenz (EFV), an activator of CYP46A1 with BBB penetration, inhibited GBM growth in vivo. CONCLUSION Our findings demonstrate that CYP46A1 is a critical regulator of cellular cholesterol in GBM and that the CYP46A1/24OHC axis is a potential therapeutic target.

BACKGROUND Tumor cells display higher cholesterol levels compared to normal cells and emerging evidence indicates a pivotal role of cholesterol content in tumor cell proliferation and growth mediated by a dysregulation of key regulators involved in cholesterol homeostasis has been implicated in various cancers. In this study, we assessed the dependency of glioblastoma (GBM) cells to extracellular cholesterol levels for growth. Through analysis of whole-transcriptome databases, we identified the cholesterol homeostasis-related genes whose expression are dysregulated in gliomas. One of the most dramatically down-regulated genes identified encodes cholesterol 24-hydroxylase (CYP46A1), a brain-specific enzyme responsible for elimination of cholesterol via conversion of cholesterol to 24(S)-hydroxycholesterol (24OHC). METHODS Molecular and clinical data was obtained from publicly genomic databases. Immunohistochemistry was applied to assess the protein level of CYP46A1 in tissue microarrays. Functionally, CYP46A1 overexpression was determined using lentiviral constructs in LN229 and a primary glioblastoma cell line (GBM#P3). Cells were characterized in vitro and implanted in vivo to generate orthotopic xenografts in order to assess their molecular status, cell survival, and self-renewal by western blotting, flow cytometry, and extreme limiting dilution assay (ELDA), respectively. RESULTS Down-regulation of CYP46A1 was found in malignant glioma samples compared to normal brain tissue. Importantly, reduced CYP46A1 expression was associated with aggressive clinicopathological features and poor prognosis of GBM patients. In functional assays, restoration of CYP46A1 suppressed cell proliferation and in vivo tumour growth by increasing 24OHC levels. Consistent, treatment of GBM cells with 24OHC inhibited tumour growth and induced apoptosis in a concentration dependent manner. RNA-seq data showed that 24OHC regulated LXR and sterol regulatory-element binding protein 1 (SREBP1) signalling activity. Intriguingly, efavirenz (EFV), a widespread used anti-HIV medication that effectively penetrates the blood–brain barrier, has recently been reported as a potent activator of the CYP46A1 enzyme. The therapeutic potential of EFV is currently being assessed in vivo and the data will be presented. CONCLUSIONS Collectively, our findings demonstrate that CYP46A1 is a critical cellular cholesterol regulator in GBM and that the CYP46A1/24OHC axis is a potential therapeutic target in GBM. Citation Format: Mingzhi Han, Shuai Wang, Xingang Li, Jian Wang, Rolf Bjerkvig. Therapeutic implications of altered cholesterol homeostasis mediated by loss of CYP46A1 in human glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-295.

Survivin has been widely reported to play a role in diagnosis and prognosis of bladder cancer patients. However, published data on this subject are heterogeneous. Here, we conducted a meta-analysis to obtain a complete evaluation of the association between survivin and recurrence-free survival (RFS), disease-specific survival (DSS), overall survival (OS), and odds ratio (OR) in bladder cancer patients. Published studies on this subject were selected for further assessment by online articles in PubMed, MEDLINE, EMBASE, and OVID databases. Pooled hazard ratios (HR) with 95 % confidence interval (95 % CI) were estimated. Funnel plots were used to evaluate the publication bias. As well, heterogeneity and sensitivity were analyzed. In this meta-analysis, we included 13 studies with the total number of 1,963 patients. Positive survivin expression in bladder cancer was associated with a poor RFS (HR, 1.831; 95 % CI, 1.344–2.49), DSS (HR, 1.721; 95 % CI, 1.477–2.004), or OS (HR, 1.753; 95 % CI, 1.092–2.816) in patients. In addition, a significant association between expression of survivin and age (OR, 0.641; 95 % CI, 0.416–0.987) as well as stage (OR, 0.37; 95 % CI, 0.190–0.750) was revealed. Heterogeneity was observed among the included studies with RFS (x 2 = 29.58, p = 0.009, I 2 = 52.7 %), OS (x 2 = 15.67, p = 0.008, I 2 = 68.1 %), and stage (x 2 = 11.97, p = 0.035, I 2 = 58.2 %). There was no publication bias according to Begg’s and Egger’s tests except for studies with gender. Furthermore, sensitivity analysis obtained the source of heterogeneity and confirmed opposite results of some studies. This study suggests that expression of survivin indicates poor prognosis in older patients and muscle invasive or advanced stage in bladder cancer. Survivin expression could be used in identifying a subgroup of patients with potential to benefit from a targeted therapy against survivin.

Up to now, survivin has been recommended as a prognostic and diagnostic indicator in glioma patients. However, there are still many controversies. Here, a meta-analysis was conducted to draw a more definitive conclusion on the correlation of survivin with overall survival (OS), age, gender, and WHO grade. Eligible studies were available through careful assessment, and then pooled hazard ratios (HRs) or odds ratios (ORs) with 95 % confidence intervals (95 % CIs) were estimated. Funnel plots were introduced to evaluate the publication bias. Additionally, heterogeneity and sensitivity were also evaluated. In the present meta-analysis, 15 eligible studies with a total of 1,089 patients were incorporated. Survivin expression in gliomas correlated with 2-year OS (n = 8; HR 0.17, 95 % CI 0.11–0.26) and 5-year OS (n = 7; HR 0.12, 95 % CI 0.07–0.22) in patients. In addition, a fixed-effect model revealed a significant association between survivin and age (male/+; OR 2.10, 95 % CI 1.44–3.05) and survivin and WHO grade (I+II/+; OR 0.27, 95 % CI 0.19–0.38). No heterogeneity was observed across all studies. According to Begg’s and Egger’s test and funnel plot, no publication bias was reported. Taken together, our meta-analysis suggests that survivin expression is associated with poor survival, older age, and higher WHO grade and could be suggested as a useful prognostic and diagnostic biomarker, or an effective therapy target.

Malignant glioma is notorious for its aggressiveness and poor prognosis, and the invasiveness of glioma cells is the major obstacle. Accumulating evidence indicates that kinesin superfamily proteins (KIFs) may play key roles in tumor invasiveness, but the mechanisms remained unresolved. Our previous study demonstrated that membrane type 1-matrix metalloproteinase (MT1-MMP) was involved in Kinesin family member 1B (KIF1B)-modulated invasion of gastric cancer cells. Therefore, the role of KIF1B in glioma cell invasion and its relationship with MT1-MMP were explored in the present study. We found that aberrantly increased expression of KIF1B was associated with worse WHO pathological classification and Karnofsky performance status (KPS), which also showed a trend towards worse prognosis. In the transwell assay, knockdown of KIF1B using siRNA repressed U87MG and A172 glioma cell migration and invasion. Silencing KIF1B inhibited expression of membranal MT1-MMP; however, the amount of MT1-MMP in the whole cell lysate was not affected. In conclusion, targeting KIF1B may be an option for anti-invasive therapies targeting glioma.

In recent years, CD133 has been identified as a cancer stem cell (CSC) marker in gliomas. Nevertheless, the clinical and prognostic value of CD133 in glioma patients remains controversial. Therefore, we conducted a systematic meta-analysis to evaluate the correlation of CD133 with World Health Organization (WHO) grade, age, gender, overall survival (OS), and progression-free survival (PFS) in glioma patients. Eligible studies on this subject were included, and then pooled odd ratios (ORs) and hazard ratios (HRs) with 95 % confidence intervals (95 % CIs) were estimated. Publication bias was assessed by the funnel plots, and heterogeneity and sensitivity were analyzed as well. In the present study, 21 articles with the total number of 1535 patients were included. High expression of CD133 in glioma patients was associated with high WHO grade (III + IV) (n = 11, OR 5.10, 95 % CI 2.99–8.69; p = 0.000), rather than age (n = 4, OR 2.54, 95 % CI 0.68–9.52; p = 0.167) and gender (n = 4, OR 0.71, 95 % CI 0.21–2.45; p = 0.587). In addition, survival analysis demonstrated a significant association between CD133 high expression and poor 2-year OS (n = 11, HR 2.18, 95 % CI 1.29–3.7; p = 0.004), 5-year OS (n = 4, HR 10.39, 95 % CI 2.59–41.63; p = 0.001), as well as PFS (n = 10, HR 2.34, 95 % CI 1.62–3.37; p = 0.000). Taken together, this study suggests that CD133 expression correlates to higher grade of gliomas and worse prognosis in glioma patients. Thus, CD133 could be recommended as a useful pathological and prognostic biomarker in clinical practice.