Autocrine/paracrine sphingosine‐1‐phosphate fuels proliferative and stemness qualities of glioblastoma stem cells

Accumulating reports suggest that human glioblastoma contains glioma stem‐like cells (GSCs) which act as key determinants driving tumor growth, angiogenesis, and contributing to therapeutic resistance. The proliferative signals involved in GSC proliferation and progression remain unclear. Using GSC lines derived from human glioblastoma specimens with different proliferative index and stemness marker expression, we assessed the hypothesis that sphingosine‐1‐phosphate (S1P) affects the proliferative and stemness properties of GSCs. The results of metabolic studies demonstrated that GSCs rapidly consume newly synthesized ceramide, and export S1P in the extracellular environment, both processes being enhanced in the cells exhibiting high proliferative index and stemness markers. Extracellular S1P levels reached nM concentrations in response to increased extracellular sphingosine. In addition, the presence of EGF and bFGF potentiated the constitutive capacity of GSCs to rapidly secrete newly synthesized S1P, suggesting that cooperation between S1P and these growth factors is of central importance in the maintenance and proliferation of GSCs. We also report for the first time that S1P is able to act as a proliferative and pro‐stemness autocrine factor for GSCs, promoting both their cell cycle progression and stemness phenotypic profile. These results suggest for the first time that the GSC population is critically modulated by microenvironmental S1P, this bioactive lipid acting as an autocrine signal to maintain a pro‐stemness environment and favoring GSC proliferation, survival and stem properties. GLIA 2014;62:1968–1981     

敲低WTAP表达抑制脑胶质瘤干细胞的增殖、迁移和侵袭

目的 探讨敲低Wilms肿瘤抑制因子（WTAP）表达对脑胶质瘤干细胞增殖、迁移和侵袭能力的影响及机制。方法 从手术切除胶质瘤标本中提取胶质瘤干细胞（GSC），应用lipofectamine2000转染WTAP shRNA质粒敲低WTAP表达，以shRNA为阴性对照；PCR检测WTAP mRNA水平分析敲低效率；MTT法检测细胞增殖能力，Transwell小室法检测细胞迁移和侵袭能力，免疫印迹法检测细胞AKT磷酸化水平。结果 从胶质瘤标本中成功提取GSC，其SOX2表达显著降低（P<0.001），而α-SMA和PDGFRb的表达上调（P<0.001）。与阴性对照组相比，WTAP敲低组GSC细胞WTAP mRNA水平明显降低（P<0.05），细胞增殖、迁移和侵袭能力均显著下降（P<0.05），细胞AKT的磷酸化水平显著降低（P<0.05）。结论 敲低WTAP表达可抑制GSC的增殖、迁移和侵袭能力，其机制可能与抑制AKT磷酸化有关。     

G-protein coupled receptor kinase (GRK)-5 regulates proliferation of glioblastoma-derived stem cells

Glioblastoma multiforme (GBM) is a grade IV malignant brain tumor with high mortality and has been well known to involve many molecular pathways, including G-protein coupled receptor (GPCR)-mediated signaling (such as epithelial growth factor receptor [EGFR] and platelet derived growth factor receptor [PDGFR]). G protein-coupled receptor kinases (GRK) directly regulate GPCR activity by phosphorylating activated agonist-bound receptors to desensitize signaling and internalize receptors through beta-arrestins. Recent studies in various cancers, including prostate and breast cancer, have highlighted the role of change in GRK expression to oncogenesis and tumor proliferation. In this study, we evaluated the expression of GRK5 in grade II to grade IV glioma specimens using immunohistochemistry and found that GRK5 expression levels are highly correlated with aggressiveness of glioma. We used culture conditions to selectively promote the growth of either glioblastoma cells with stem cell markers (GSC) or differentiated glioblastoma cells (DGC) from fresh GBM specimens. GSC are known to be highly invasive and mobile, and have the capacity to self-renew and are more resistant to chemotherapy and radiation compared to differentiated populations of GBM. We examined the expression of GRK5 in these two sets of culturing conditions for GBM cells and found that GRK5 expression is upregulated in GSC compared to differentiated GBM cells. To better understand the role of GRK5 in GBM-derived stem cells, we created stable GRK5 knockdown and evaluated the proliferation rate. Using an ATP chemiluminescence assay, we show, for the first time, that knocking down the expression of GRK5 decreased the proliferation rate of GSC in contrast to control.     

Transdifferentiation of glioblastoma stem-like cells into mural cells drives vasculogenic mimicry in glioblastomas

Recent evidence has shown that glioblastoma stem-like cells (GSCs) can transdifferentiate into endothelial cells and vascular-like tumor cells. The latter pattern of vascularization indicates an alternative microvascular circulation known as vasculogenic mimicry (VM). However, it remains to be clarified how the GSC-driven VM makes a significant contribution to tumor vasculature. Here, we investigated 11 cases of glioblastomas and found that most of them consisted of blood-perfused vascular channels that coexpress mural cell markers smooth muscle α-actin and platelet-derived growth factor receptor β, epidermal growth factor receptor, and vascular endothelial growth factor receptor 2 (Flk-1), but not CD31 or VE-cadherin. This microvasculature coexisted with endothelial cell-associated vessels. GSCs derived from patients with glioblastomas developed vigorous mural cell-associated vascular channels but few endothelial cell vessels in orthotopic animal models. Suppression of Flk-1 activity and gene expression abrogated GSC transdifferentiation and vascularization in vitro, and inhibited VM in animal models. This study establishes mural-like tumor cells differentiated from GSCs as a significant contributor to microvasculature of glioblastoma and points to Flk-1 as a potential target for therapeutic intervention that could complement current anti-angiogenic treatment.     

A molecularly distinct subset of glioblastoma requires serum-containing media to establish sustainable bona fide glioblastoma stem cell cultures

Glioblastoma (GBM) is the most frequent and deadly primary malignant brain tumor. Hallmarks are extensive intra-tumor and inter-tumor heterogeneity and highly invasive growth, which provide great challenges for treatment. Efficient therapy is lacking and the majority of patients survive less than 1 year from diagnosis. GBM progression and recurrence is caused by treatment-resistant glioblastoma stem cells (GSCs). GSC cultures are considered important models in target identification and drug screening studies. The current state-of-the-art method, to isolate and maintain GSC cultures that faithfully mimic the primary tumor, is to use serum-free (SF) media conditions developed for neural stem cells (NSCs). Here we have investigated the outcome of explanting 218 consecutively collected GBM patient samples under both SF and standard, serum-containing media conditions. The frequency of maintainable SF cultures (SFCs) was most successful, but for a subgroup of GBM specimens, a viable culture could only be established in serum-containing media, called exclusive serum culture (ESC). ESCs expressed nestin and SOX2, and displayed all functional characteristics of a GSC, that is, extended proliferation, sustained self-renewal and orthotopic tumor initiation. Once adapted to the in vitro milieu they were also sustainable in SF media. Molecular analyses showed that ESCs formed a discrete group that was most related to the mesenchymal GBM subtype. This distinct subgroup of GBM that would have evaded modeling in SF conditions only provide unique cell models of GBM inter-tumor heterogeneity.     

Type 1 collagen as a potential niche component for CD133‐positive glioblastoma cells

Cancer stem cells are thought to be closely related to tumor progression and recurrence, making them attractive therapeutic targets. Stem cells of various tissues exist within niches maintaining their stemness. Glioblastoma stem cells (GSCs) are located at tumor capillaries and the perivascular niche, which are considered to have an important role in maintaining GSCs. There were some extracellular matrices (ECM) on the perivascular connective tissue, including type 1 collagen. We here evaluated whether type 1 collagen has a potential niche for GSCs. Imunohistochemical staining of type 1 collagen and CD133, one of the GSCs markers, on glioblastoma (GBM) tissues showed CD133‐positive cells were located in immediate proximity to type 1 collagen around tumor vessels. We cultured human GBM cell lines, U87MG and GBM cells obtained from fresh surgical tissues, T472 and T555, with serum‐containing medium (SCM) or serum‐free medium with some growth factors (SFM) and in non‐coated (Non‐coat) or type 1 collagen‐coated plates (Col). The RNA expression levels of CD133 and Nestin as stem cell markers in each condition were examined. The Col condition not only with SFM but SCM made GBM cells more enhanced in RNA expression of CD133, compared to Non‐coat/SCM. Semi‐quantitative measurement of CD133‐positive cells by immunocytochemistry showed a statistically significant increase of CD133‐positive cells in Col/SFM. In addition, T472 cell line cultured in the Col/SFM had capabilities of sphere formation and tumorigenesis. Type 1 collagen was found in the perivascular area and showed a possibility to maintain GSCs. These findings suggest that type 1 collagen could be one important niche component for CD133‐positive GSCs and maintain GSCs in adherent culture.     

肿瘤微环境对胶质瘤干细胞影响机制的研究进展

胶质瘤干细胞（GSCs）是具有干性特征和功能的胶质瘤细胞,近些年来受到广泛关注。与普通胶质瘤细胞相比,GSCs具有很强的成瘤、侵袭、化疗放疗抵抗能力,可能是导致转移和肿瘤复发的主要因素。随着研究不断深入,人们逐渐发现肿瘤微环境（TME）在GSCs的形成中发挥重要作用,并且TME能够促进GSCs表型的维持。该文将围绕TME对GSCs的影响和机制的前沿进展进行讨论;并详细介绍TME中缺氧、酸性、损伤相关分子模式（DAMP）如高迁移率族蛋白B1（HMGB1）以及外泌体对GSCs的具体作用及机制。     

High expression of RFX4 is associated with tumor progression and poor prognosis in patients with glioblastoma

Abstract

Aim: Glioma stem cells (GSCs) have been shown to contribute to tumor development and recurrence, therapeutic resistance, and cellular heterogeneity of glioblastoma multiforme (GBM). Recently, it has been reported that GSCs lose their self-renewal ability and tumorigenic potential upon differentiation. In this study, we identified Regulatory Factor X4 (RFX4) gene to regulate GSCs’ survival and self-renewal activity in the GBM patients samples.

Materials and methods: We utilized public datasets from the Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Ivy Glioblastoma Atlas Project, and The Human Protein Atlas to screen candidate genes which are associated with the development of GBM and poor patients survival. Small hairpin RNA (shRNA) lentivirus was applied to knockdown RFX4 gene in GSCs.

Results: We found that RFX4 mRNA expression among the RFX family was particularly reduced during GSC differentiation. RT-qPCR analysis revealed significant downregulation of RFX4 and stem cell markers (CD15 and CD133) mRNA expressions in primary human GBM-derived GSCs cultured under serum condition. Consistently, GSCs showed significantly elevated RFX4 mRNA expression levels compared to normal astrocytes, NHA, whereas glioma cells did not. Furthermore, analysis of the TCGA data set revealed that RFX4 is highly expressed in GBM, and contributes to the lowering of patient survival. Depletion of RFX4 using shRNA lentivirus in patient GBM-derived GSCs decreased neurosphere formation and cell viability.

Conclusion: These results suggest that RFX4 is a potential risk factor for maintaining the stemness of GSCs and making glioma more malignant, and thus, could be a promising target of GBM treatment.     

miR-145 Inhibits Migration and Invasion of Glioma Stem Cells by Targeting ABCG2

Despite advances in clinical therapies and technologies, the prognosis for patients with malignant glioma is poor. Our previous research demonstrated that glioma stem cells (GSCs) were crucial for glioma malignancy and accelerated tumor migration and invasion. The migration and invasion of malignant glioma cells into the surrounding normal brain tissues cause the poor outcome. miR-145, a miRNA found to be expressed in neurons, was recently found to have reduced expression in glioblastoma multiforme tumors. And miR-145 loss in glioma cells led to increased cell proliferation and invasion. However, its function on the migration and invasion of GSCs was still unknown. In this study, we aimed to identify the effects and mechanisms of miR-145 on the migration and invasion of GSCs. Our investigations revealed that miR-145 was low expressed in malignant glioma tissues and their corresponding GSCs. Knockdown of miR-145 in vitro could enhance the migration and invasion of GSCs, while up-regulation of miR-145 had the opposite effects. Further investigation of the potential mechanism demonstrated that the function of miR-145 in regulating the migration and invasion of GSCs is mediated by its targeting of ABCG2 mRNA. ABCG2 is an ATP-binding cassette transporter protein, which was identified to be overexpressed in GSCs and higher-grade glioma tissues. We found that miR-145 was negative correlated with ABCG2 levels in GSCs, and reduction in ABCG2 expression decreased the cell migration and invasion of GSCs. Further, a luciferase reporter proved that ABCG2 was a direct target of miR-145 in GSCs. Thus, these findings underscore the potential of miR-145 to regulate the migration and invasion of GSCs through targeting ABCG2.     

人胶质瘤干细胞内在自我更新能力

目的 了解胶质瘤干细胞内在的自我更新和增殖能力。方法 观察原代胶质瘤细胞在单纯改良Eagle/F12培养液（DMEM/F12）中胶质瘤干细胞球的形成,并检测其CD133、胶质纤维酸性蛋白（GFAP）、微管相关蛋白（MAP2）、髓磷脂碱性蛋白（MBP）的表达。通过二代球体形成、细胞增殖测定、分化实验分析其自我更新、增殖、多能分化能力。通过裸鼠移植瘤实验观察所分离细胞球细胞与原代培养胶质瘤细胞成瘤能力的差异。结果 在单纯DMEM/F12培养液中形成的胶质瘤细胞球细胞表达神经干细胞标记CD133,不表达分化标志GFAP、MAP2,少数细胞表达MBP。分离出的胶质瘤细胞球细胞可在单纯DMEM/F12培养基中增殖,并能形成CD133阳性的二代细胞球,可分化为GFAP、MAP2、MBP阳性表达的肿瘤细胞。裸鼠成瘤实验显示其成瘤能力显著高于原代胶质瘤细胞。结论 胶质瘤干细胞能在无血清、无外源性细胞因子培养基中形成肿瘤干细胞球,胶质瘤干细胞的自我更新和增殖不依赖于外源性生长因子,它可能拥有自我更新的自身活化机制。     

An overview of crucial genes involved in stemness of glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common and first leading cause of death in primary human brain cancers. Following current interventions, despite continuous advancements in medicine, the median survival time of patients is unfavorable regardless of radiotherapy, chemotherapy, or surgery. This poor outcome is referred to “cancer stem cell” (CSC) concept. According to it a minority of cells within the GBM mass originates from neural stem cells (NSCs) or alternatively hijack similar checkpoints in them that evolution has selected in order to prevent inappropriate neurogenic-to-gliogenic switch which is responsible for the growth of tumor and its resistance to existing therapies. New developments in non-surgical treatment of GBM are based on this knowledge, however, similarities in molecular pathways between GBM and glioma stem cells (GSCs) motive the race to identify and target GSC regulators with less and more harmful effect in normal adult NSC and GBM respectively. In it the implications of downstream stemness genes with modulatory roles for stemness state pathways are assumed importance. This review, embodies NSC-intrinsic genes by recent elucidated roles of importance for GBM to affix to the stemness. Therefor it could facilitate a holistic approach of molecular targeted therapy to compromise the formidable resistant GBM to eradication.     

阿苯达唑抑制胶质瘤裸鼠模型肿瘤生长

目的 探讨阿苯达唑（ABZ）对胶质瘤裸鼠模型肿瘤生长的影响。方法 将术中获取的胶质母细胞组织消化为单细胞悬液用无血清干细胞培养基进行培养胶质母细胞瘤干细胞（GSC）,用含10%胎牛血清的DMEM培养基培养胶质瘤细胞系U87、U251、U172,以25、50、100、200 ng/ml的终浓度ABZ作用细胞,采用MTT法检测细胞增殖。右侧腋窝皮下注射0.2 ml（5×106个细胞）GSC及U87细胞悬液构建胶质瘤裸鼠模型,将30只胶质瘤裸鼠模型随机分为6组,GSC和U87细胞移植各3组,每种移植瘤模型均分为模型组（腹腔注射等体积DMSO）、低剂量ABZ组（腹腔注射ABZ,50 mg/kg）、高剂量ABZ组（腹腔注射ABZ,100 mg/kg）。定时测量肿瘤长径与短径,计算肿瘤体积;PCR法和免疫印迹法检测裸鼠肿瘤组织血管内皮生长因子（VEGF）mRNA和蛋白表达水平。结果 ABZ对GSC、U87、U251和A172细胞生长均具有明显抑制效果（P<0.05）,而且抑制效果具有浓度依赖性（P<0.05）,浓度>50 ng/ml抑制效果较好。腹腔注射ABZ后,高剂量和低剂量ABZ组移植瘤体积增长较对照组均明显减慢（P<0.05）。高剂量ABZ组和低剂量ABZ组肿瘤VEGF mRNA和蛋白表达水平较对照组均明显降低（P<0.05）。结论 ABZ可以抑制胶质瘤裸鼠模型肿瘤生长,可能与抑制肿瘤细胞增殖和血管生成有关。     

Downregulation of ADAMTS3 Suppresses Stemness and Tumorigenicity in Glioma Stem Cell

Aims

Glioblastoma multiforme (GBM) is the most aggressive type of human brain tumor, with a poor prognosis and a median overall survival of fewer than 15 months. Glioma stem cells (GSCs) have recently been identified as a key player in tumor initiation and therapeutic resistance in GBM. ADAMTS family of metalloproteinases is known to cleave a wide range of extracellular matrix substrates and has been linked to tissue remodeling events in tumor development. Here, we investigate that ADAMTS3 regulates GSC proliferation and self-renewal activities, and tumorigenesis in orthotopic xenograft models.

Methods

ADAMTS3 mRNA expression levels in normal human astrocyte (NHA), glioma, and GSCs cell lines were compared. After knockdown of ADAMTS3, alamarBlue assay, in vitro limiting dilution, and orthotopic xenograft assays were performed. To investigate the tumor-associated roles of ADAMTS3, several statistical assays were conducted using publicly available datasets.

Results

ADAMTS3 level was remarkably higher in GSCs than in NHA, glioma cell lines, and their matched differentiated tumor cells. Interestingly, knockdown of ADAMTS3 disrupted GSC's proliferation, self-renewal activity, and tumor formation in vivo. Furthermore, ADAMTS3 could be used as an independent predictor of malignancy progression in GBM.

Conclusion

We identified ADAMTS3 as a potential therapeutic target for GBM.     

CD133+ glioblastoma stem-like cells induce vascular mimicry in vivo

Glioblastoma is one of the most angiogenic malignancy, the neoplastic vessels of which are likely to arise by angiogenesis and vasculogenesis. An alternative mechanism of tumor vasculature is described, termed vasculogenic mimicry, by which highly aggressive tumor cells can form vessel-like structures themselves, by virtue of their high cellular plasticity. Evidence suggests that cancer stem cells acquire a multi-potent plastic phenotype and show vasculogenic potential. In this study, we report that glioblastoma stem-like cells (GSCs) can form vasculogenic mimicry in tumor xenografts and express pro-vascular molecules. We isolated GSCs from resected human glioblastoma tissues and demonstrated their stemness, differentiation, and in vivo tumor-initiating potential. Through a limiting dilution assay, CD133+ (CD133(+)-GSC) and CD133- (CD133(-)-GSC) subpopulation of GSCs were obtained. Orthotopic xenotransplantation study revealed that these two subpopulations of GSCs shared similar efficacy in tumor formation but showed distinct intratumor vasculature. In comparison with CD133(-)-GSC, a highly vascularized anaplastic tumor, mimicking vasculogenic mimicry, was found in CD133(+)-GSC-derived tumor xenografts. Subsets of CD133(+)-GSC but not CD133(-)-GSC were capable of vascular smooth muscle-like cell differentiation, in vitro and in vivo. In tumor xenografts, endothelium-associated CD31 gene was detected in implanted CD133(-)-GSC and exclusively dispersed within the tumor tissues. Although the detailed action mechanisms required further investigation, this study demonstrated the vasculogenic capacity of brain GSCs and their cellular plasticity. The results of expression of pro-vascular molecules and differentiation of vascular-like cells suggest that GSCs may contribute to form vessel-like structures and provide a blood supply for glioblastoma cells.     

MiR-29a-Mediated CD133 Expression Contributes to Cisplatin Resistance in CD133<Superscript>+</Superscript> Glioblastoma Stem Cells

CD133 positive (CD133⁺) cells are cancer stem cells in glioblastoma that are associated with poor prognosis and resistance to radiotherapy. However, the role of CD133 in chemoresistance is inconclusive, although recent studies suggest that increased CD133 expression may lead to increased cisplatin resistance under certain circumstances. In this study, we further explored the mechanism underlying CD133-mediated cisplatin resistance in glioblastoma stem cells. We sorted human glioblastoma T98G and U87MG cells into CD133⁺ and CD133^? pools and measured apoptosis and CD133 expression levels in response to cisplatin treatment. We predicted candidate microRNAs that might target CD133 and assessed their levels in cisplatin-treated CD133⁺ cells. Finally, we overexpressed miR-29a in CD133⁺ cells and tested its effects in cisplatin-mediated apoptosis and survival of CD133⁺ tumor bearing mice receiving cisplatin treatment. We found that CD133⁺ glioblastoma stem cells showed more resistance to cisplatin treatment. Cisplatin increased CD133 expression by suppressing miR-29a levels. MiR-29a overexpression improved sensitivity of cisplatin in CD133⁺ cells and significantly suppressed tumor growth in CD133⁺ tumor bearing mice in response to cisplatin treatment. Our data show that miR-29a ameliorates CD133-mediated chemoresistance in glioblastoma stem cells, suggesting it as a potential therapeutic target for treating glioblastoma.     

Enhancer of Zeste 2 (EZH2) is up-regulated in malignant gliomas and in glioma stem-like cells

F. Orzan, S. Pellegatta, P. L. Poliani, F. Pisati, V. Caldera, F. Menghi, D. Kapetis, C. Marras, D. Schiffer and G. Finocchiaro (2011) Neuropathology and Applied Neurobiology 37, 381–394
Enhancer of Zeste 2 (EZH2) is up‐regulated in malignant gliomas and in glioma stem‐like cells Aims: Proteins of the Polycomb repressive complex 2 (PRC2) are epigenetic gene silencers and are involved in tumour development. Their oncogenic function might be associated with their role in stem cell maintenance. The histone methyltransferase Enhancer of Zeste 2 (EZH2) is a key member of PRC2 function: we have investigated its expression and function in gliomas. Methods: EZH2 expression was studied in grade II–IV gliomas and in glioma stem‐like cells (GSC) by quantitative PCR and immunohistochemistry. Effects of EZH2 down‐regulation were analysed by treating GSC with the histone deacetylase (HDAC) inhibitor suberoylanide hydroxamic acid (SAHA) and by shRNA. Results: DNA microarray analysis showed that EZH2 is highly expressed in murine and human GSC. Real‐time PCR on gliomas of different grade (n = 66) indicated that EZH2 is more expressed in glioblastoma multiforme (GBM) than in low‐grade gliomas (P = 0.0013). This was confirmed by immunohistochemistry on an independent set of 106 gliomas. Treatment with SAHA caused significant up‐regulation of PRC2 predicted target genes, GSC disruption and decreased expression of EZH2 and of the stem cell marker CD133. Inhibition of EZH2 expression by shRNA was associated with a significant decrease of glioma proliferation. Conclusion: The data suggest that EZH2 plays a role in glioma progression and encourage the therapeutic targeting of these malignancies by HDAC inhibitors.