Xing-gang Mao

CDH5 is specifically activated in glioblastoma stemlike cells and contributes to vasculogenic mimicry induced by hypoxia

BACKGROUND A proportion of glioblastoma stemlike cells (GSCs) expressing endothelial cell marker CDH5 (vascular-endothelial-cadherin or CD144) can transdifferentiate into endothelial cells and form blood vessels. However, the implications of CDH5 expression in gliomas and how it is regulated in GSCs remain to be clarified. METHODS The mRNA and protein levels of CDH5 were detected in glioma samples and cultured cell lines, and the prognostic value of the CDH5 expression level for GBM patients was evaluated. Bioinformatics analysis was performed to reveal the potential functional roles of CDH5 in glioblastoma multiforme. Gene knockdown induced by short hairpin RNA, chromatin immunoprecipitation analysis, and a vasculogenic tube formation assay were performed to investigate the relationships among hypoxia, CDH5 expression level, and angiogenesis. RESULTS CDH5 was overexpressed in gliomas, correlated with tumor grades, and was an independent adverse prognostic predictor for glioblastoma multiforme patients. CDH5 was specifically activated in GSCs but not in non-GSCs or neural stem cells, and CDH5(+) cells could produce xenografts in immunocompromised mice. Bioinformatics analysis demonstrated that CDH5 might interact directly with hypoxia-inducible factor (HIF)2α. CDH5 expression was significantly upregulated in GSCs, but not in non-GSCs or normal neural stem cells, under a 1% O2 condition. Both HIF1α and HIF2α positively regulated CDH5 level in GSCs and could bind to the promoter of CDH5. Furthermore, CDH5 contributed to the vasculogenic mimicry of GSCs, especially under hypoxic conditions. CONCLUSIONS The specific expression of CDH5 in GSCs may contribute to GSC-derived neovasculogenesis in glioblastoma multiforme, especially under hypoxic conditions, revealing novel tumorigenic mechanisms contributed by GSCs.

Overexpression of ZNF217 in glioblastoma contributes to the maintenance of glioma stem cells regulated by hypoxia-inducible factors.

Glioblastoma multiforme (GBM) is the most aggressive and common kind of primary brain tumor in adults, and is thought to be driven by a subpopulation of glioma stem cells (GSCs). GSCs reside in a specialized hypoxic niche, which can regulate the tumorigenic capacity of GSCs primarily through the hypoxia-inducible factors (HIFs), HIF1α and HIF2α. ZNF217 is an oncogene frequently amplified in many kinds of tumors. It is associated with aggressive tumor behavior and poor clinical prognosis, but its role in gliomas is poorly known. Gene expression and copy number analysis from TCGA data reveal that ZNF217 is amplified in 32% and overexpressed in 71.2% of GBMs. Quantitative RT–PCR and western blotting of a cohort of glioma samples showed that ZNF217 was highly expressed in gliomas and increased with tumor grade. Analysis of a molecular database demonstrated that ZNF217 expression correlated with poor survival of glioma patients. Investigation of ZNF217 expression in GSCs, non-GSCs and normal neural stem cells (NSCs) indicated that ZNF217 was more highly expressed in GSCs than in non-GSCs and NSCs. Knockdown of ZNF217 in GSCs by small-interfering RNA (siRNA) inhibited their growth and promoted their differentiation. Interestingly, ZNF217 was upregulated in GSCs and the GBM cell line U87 when exposed to the hypoxic environment of 1% oxygen. Knockdown of either HIF1α or HIF2α, which has a central role in the hypoxia-induced responses of these cells, inhibited ZNF217 expression. In addition, ZNF217 upregulation was compromised under hypoxia in U87 and GSCs when either HIF1α or HIF2α was targeted by siRNA. HIF2α knockdown inhibited ZNF217 expression more efficiently in both normoxia and hypoxia than HIF1α knockdown. Therefore, ZNF217 is overexpressed in GBMs and contributes to the maintenance of GSCs, which is regulated by HIFs released by the hypoxic environment of the tumor.

Knockdown of FRAT1 expression by RNA interference inhibits human glioblastoma cell growth, migration and invasion.

Background FRAT1 positively regulates the Wnt/β-catenin signaling pathway by inhibiting GSK-3-mediated phosphorylation of β-catenin. It was originally characterized as a protein frequently rearranged in advanced T cell lymphoma, but has recently also been identified as a proto-oncogene involved in tumorigenesis. Our previous studies showed that FRAT1 was dramatically overexpressed in gliomas and its expression level was significantly increased along with clinicopathological grades. Methods In the current study, we used RT-PCR and Western blotting to assess the mRNA and protein levels of FRAT1 in three glioma cell lines. In addition, to evaluate its functional role in gliomas, we examined the effects of FRAT1 knockdown on proliferation, migration and invasion in vitro and tumor growth in vivo using glioblastoma U251 cells and RNAi. Results FRAT1 was highly expressed in all three glioma cell lines. RNAi-mediated down-regulation of endogenous FRAT1 in human glioblastoma U251 cells resulted in suppression of cell proliferation, arrest of cell cycle, inhibition of cell migration and invasion in vitro. Moreover, FRAT1 depletion significantly impaired tumor xenograft growth in nude mice. Conclusions Our results highlight the potential role of FRAT1 in tumorigenesis and progression of glioblastoma. These findings provide a biological basis for FRAT1 as a potential molecular marker for improved pathological grading and as a novel candidate therapeutic target for glioblastoma management.

The expression profile of FRAT1 in human gliomas

FRAT1 was originally characterized as a protein frequently rearranged in advanced T cell lymphoma, which inhibits GSK-3-mediated phosphorylation of beta-catenin and positively regulates the Wnt signaling pathway. FRAT1 has been identified as a proto-oncogene involved in tumorigenesis. Previous studies have shown that FRAT1 is strikingly overexpressed in some human cancers. However, the relationship between FRAT1 and human gliomas is unclear. In this study, we detected the expression of FRAT1 in human gliomas by immunohistochemistry, Western blot and RT-PCR. FRAT1 was found to be specifically expressed in the majority of glioma samples, and their expression levels increased markedly with the increase of WHO grades. In addition, there was a positive correlation between FRAT1 immunoreactivity score (IRS) and beta-catenin IRS. Our results suggest that FRAT1 may be an important factor in the tumorigenesis and progression of gliomas, and could be used as a potential molecular marker for pathological diagnosis and a target for biological therapy.

Progress on Potential Strategies to Target Brain Tumor Stem Cells

The identification of brain tumor stem cells (BTSCs) leads to promising progress on brain tumor treatment. For some brain tumors, BTSCs are the driving force of tumor growth and the culprits that make tumor revive and resistant to radiotherapy and chemotherapy. Therefore, it is specifically significant to eliminate BTSCs for treatment of brain tumors. There are considerable similarities between BTSCs and normal neural stem cells (NSCs), and diverse aspects of BTSCs have been studied to find potential targets that can be manipulated to specifically eradicate BTSCs without damaging normal NSCs, including their surface makers, surrounding niche, and aberrant signaling pathways. Many strategies have been designed to kill BTSCs, and some of them have reached, or are approaching, effective therapeutic results. Here, we will focus on advantages in the issue of BTSCs and emphasize on potential therapeutic strategies targeting BTSCs.

Maintenance of Critical Properties of Brain Tumor Stem-like Cells After Cryopreservation

It would be very useful to be able to classify brain tumor stem cells (BTSCs) by certain criteria to afford the design of specific or individualized treatment. Here, we studied two BTSC lines with differing biological and molecular features and whose respective features were well preserved after cryopreservation as single cells in SFM or 90% serum with 10% DMSO, a method not previously reported. The resuscitated BTSCs shared properties indistinguishable from their respective parental cells, including tumor sphere forming potentials, growth and differentiation properties, and tumorigenesis in vivo. The two cell lines also had differing molecule profiles, which can be well preserved after cryopreservation, similar to that of their respective primary tumors. Therefore, BTSCs from different patients, that have their own properties, were well retained by the present cryopreservation method, which might be a useful and reliable method for preserving BTSCs for long-term studies, such as classification and specific therapy design.

Hypoxia upregulates HIG2 expression and contributes to bevacizumab resistance in glioblastoma

Hypoxia contributes to the maintenance of stem-like cells in glioblastoma (GBM), and activates vascular mimicry and tumor resistance to anti-angiogenesis treatments. The present study examined the expression patterns and biological significance of hypoxia-inducible protein 2 (HIG2, also known as HILPDA) in GBM. HIG2 was highly expressed in gliomas and was correlated with tumor grade, and high HIG2 expression independently predicted poor GBM patient prognosis. HIG2 was upregulated during hypoxia and by hypoxia mimics, and HIG2 knockdown in GBM cells inhibited cell proliferation and invasion. HIF1α bound to the HIG2 promoter and increased its expression in GBM cells, and HIG2 upregulated HIF1α expression. Reconstruction of a HIG2-related molecular network using bioinformatics methods revealed that HIG2 is closely correlated with angiogenesis genes, such as VEGFA, in GBM. HIG2 levels positively correlated with VEGFA in GBM samples. In addition, treatment of transplanted xenograft nude mice with bevacizumab (anti-angiogenesis therapy) resulted in HIG2 upregulation at late stages. We conclude that HIG2 is overexpressed in GBM and upregulated by hypoxia, and is a potential novel therapeutic target. HIG2 overexpression is an independent prognostic indicator and may promote tumor resistance to anti-angiogenesis treatments.

LGR5/GPR49 is implicated in motor neuron specification in nervous system.

The biological roles of stem cell marker LGR5, the receptor for the Wnt-agonistic R-spondins, for nervous system are poorly known. Bioinformatics analysis in normal human brain tissues revealed that LGR5 is closely related with neuron development and functions. Interestingly, LGR5 and its ligands R-spondins (RSPO2 and RSPO3) are specifically highly expressed in projection motor neurons in the spinal cord, brain stem and cerebral. Inhibition of Notch activity in neural stem cells (NSCs) increased the percentage of neuronal cells and promoted LGR5 expression, while activation of Notch signal decreased neuronal cells and inhibited the LGR5 expression. Furthermore, knockdown of LGR5 inhibited the expression of neuronal markers MAP2, NeuN, GAP43, SYP and CHRM3, and also reduced the expression of genes that program the identity of motor neurons, including Isl1, Lhx3, PHOX2A, TBX20 and NEUROG2. Our data demonstrated that LGR5 is highly expressed in motor neurons in nervous system and is involved in their development by regulating transcription factors that program motor neuron identity.

Overexpression of FRAT1 Is Associated with Malignant Phenotype and Poor Prognosis in Human Gliomas

Glioma is the most common malignancy of the central nervous system. Approximately 40 percent of intracranial tumors are diagnosed as gliomas. Difficulties in treatment are associated closely with the malignant phenotype, which is characterized by excessive proliferation, relentless invasion, and angiogenesis. Although the comprehensive treatment level of brain glioma is continuously progressing, the outcome of this malignancy has not been improved drastically. Therefore, the identification of new biomarkers for diagnosis and therapy of this malignancy is of significant scientific and clinical value. FRAT1 is a positive regulator of the Wnt/β-catenin signaling pathway and is overexpressed in many human tumors. In the present study, we investigated the expression status of FRAT1 in 68 patients with human gliomas and its correlation with the pathologic grade, proliferation, invasion, angiogenesis, and prognostic significance. These findings suggest that FRAT1 may be an important factor in the tumorigenesis and progression of glioma and could be explored as a potential biomarker for pathological diagnosis, an indicator for prognosis, and a target for biological therapy of malignancy.

The Potential of the Brain: Plasticity Implications for De-Differentiation of Mature Astrocytes

Neural stem cells (NSCs) have been identified for many years, mainly based on their functional properties including extensive self-renewal and multi-differentiation potential (Reynolds and Weiss 1992). NSCs express many stem cell proteins such as nestin, SOX2, SSEA1, and CD133 et al., while confident markers that can specifically recognize these cells are lacking. It has long been suggested that GFAPpositive cells in the neurogenic area, subventricular zone (SVZ), are NSCs (Doetsch et al. 1999; Doetsch 2003b; Laywell et al. 2000; Sanai et al. 2004; Merkle and AlvarezBuylla 2006), and one type of NSCs in subgranular zone (SGZ) is GFAP positive (Garcia et al. 2004). Recent studies focusing on micro-niche of SVZ NSCs further strengthened this idea, which implied GFAP cells in SVZ were genuine NSCs whose maintenance might depend on a complicated microenvironment (Doetsch 2003a; Shen et al. 2008; Tavazoie et al. 2008; Mirzadeh et al. 2008). These studies did not rule out the possibility that other mature GFAP astrocytes beyond the SVZ and SGZ are, or can transform into, NSCs. Therefore, direct evidence that mature astrocytes can transform into NSCs is scarce, and, importantly, it is not clear how to transform astrocytes, if possible, into NSCs. Seeking for the identity of NSCs and the relationship between GFAP cells and NSCs were critical for neurobiology research, for GFAP cells were the most widespread dividing cells in the central nervous system (CNS). Elucidating this issue is very important for many CNS-related diseases such as brain injury and spinal cord injury and brain tumors. Repairing the injured CNS tissues needs re-building or expanding the NSCs pools, while seeking the origin of brain tumors needs elucidating ways that brain tumor stem cells transformed from NSCs or astrocytes, which might transform into NSCs firstly. The work of Yang et al. demonstrated definitely that the mature astrocytes could transform into NSCs by a simple way—injury induced by scratch or culturing with conditioned culture medium of scratch-insulted astrocytes (Yang et al. 2009). This work continues and confirms the prior work demonstrating that the mature astrocytes may acquire NSCs potential after spinal cord injury (Lang et al. 2004). In the previous work, Lang et al. demonstrated that injury in the spinal cord of adult rats can induce de-differentiation of mature astrocytes into stem or progenitor cells (Lang et al. 2004). By using an ingenious design, they ruled out the possibility that these de-differentiated astrocytes with NSC properties were migrated from the central canal of the injured spinal cord where NSCs reside under normal conditions. Yang et al. testified in vitro that the mature astrocytes could re-acquire NSCs potentials under injury conditions and, to some degree, elucidate some critical regulatory mechanisms of this process. This study reinforced the hypothesis that GFAP mature astrocytes can transform into NSCs (Doetsch 2003b; Yu et al. 2006). Two key steps of astrocytes isolation guaranteed the astrocytes used in the study were mature astrocytes, and not contaminated undifferentiated cells. First, astrocytes were isolated from lateral spinal cord of postnatal 15 days excluding the central canal region, which precluded the immature cells during the first two postnatal weeks X. Mao X. Zhang (&) Department of Neurosurgery, Xijing Institute of Clinical Neuroscience, Xijing Hospital, The Fourth Military Medical University, 710032 Xi’an, Shannxi Province, People’s Republic of China e-mail: xzhang@fmmu.edu.cn