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. 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. 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. 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.

In recent years, a small number of cells that have stem cell properties were identified in human gliomas called brain tumor stem cells (BTSCs), which were thought to mainly contribute to the initiation and development of gliomas and could be identified by the surface marker CD133. However, recent studies indicated that the expression of CD133 might be regulated by environmental conditions such as hypoxia and that there might be CD133(-) BTSCs. Genetic mouse models demonstrated that some gliomas originated from transformed neural stem cells (NSCs). Therefore, we investigated the expression of CD15, a surface marker for NSCs, in tumor spheres derived from astrocytoma and ependymoma. CD15(+) cells isolated from these tumor spheres had properties of BTSCs including self-renewal, multidifferentiation, and the ability to recapitulate the phenocopy of primary tumors. CD15 exhibited stable expression in long-term cultured tumor spheres, which sustained BTSCs properties, whereas CD133 expression decreased significantly in late passages. Furthermore, CD15(+)CD133(-) cells isolated from early or late passages of tumor spheres showed similar characteristics of BTSCs. Examination of glioma samples by immunohistochemistry showed that CD15 was expressed in a subset of human brain tumors. Therefore, CD15 can be used as a marker of stem-like cells derived from brain tumors that might contain CD133(-) BTSCs.

Background: LAT1/4F2hc heterodimeric complex is a major route for the transport of large neutral essential amino acids through the plasma membrane. Although it has been shown that LAT1/4F2hc is highly expressed in a variety of human tumors including gliomas, and LAT1 over-expression is associated with glioma grade and poor prognosis of glioma patients, the precise tissue location of LAT1/4F2hc in gliomas and the precise role of LAT1/4F2hc in glioma biological features remain unclear. Methods: In the current study, the expressions of LAT1, 4F2hc, CD34 and Ki-67 were investigated by immunohistochemistry in 62 cases of human brain glioma; LAT1/4F2hc expression level, Ki-67 labeling index (Ki-67 LI) and microvessel density (MVD) were measured semi-quantitatively; and the correlation of LAT1/4F2hc expression with histopathological features, Ki-67 LI and MVD in gliomas was further analyzed. Results: The results showed that both LAT1 and 4F2hc were expressed in all examined specimens. LAT1 but 4F2hc expression levels significantly correlated with the pathological grade and both expression levels significantly correlated with Ki-67 LI of gliomas. We also demonstrated that both LAT1 and 4F2hc immunoreactivity were observed in tumor cells as well as vascular endothelia; furthermore, the LAT1 expression level was markedly associated with glioma MVD as well. Conclusion: LAT1/4F2hc over-expression is closely correlates with the malignant phenotype and proliferation of gliomas, and LAT1 was associates with glioma angiogenesis. LAT1/4F2hc, especially LAT1, may become a novel potential molecular target for glioma biological therapy.

Hypoxia contributes to the initiation and progression of glioblastoma by regulating a cohort of genes called hypoxia-regulated genes (HRGs) which form a complex molecular interacting network (HRG-MINW). Transcription factors (TFs) often play central roles for MINW. The key TFs for hypoxia induced reactions were explored using proteomic analysis to identify a set of hypoxia-regulated proteins (HRPs) in GBM cells. Next, systematic TF analysis identified CEBPD as a top TF that regulates the greatest number of HRPs and HRGs. Clinical sample and public database analysis revealed that CEBPD is significantly up-regulated in GBM, high levels of CEBPD predict poor prognosis. In addition, CEBPD is highly expressed in hypoxic condition both in GBM tissue and cell lines. For molecular mechanisms, HIF1α and HIF2α can activate the CEBPD promotor. In vitro and in vivo experiments demonstrated that CEBPD knockdown impaired the invasion and growth capacity of GBM cells, especially in hypoxia condition. Next, proteomic analysis identified that CEBPD target proteins are mainly involved in the EGFR/PI3K pathway and extracellular matrix (ECM) functions. WB assays revealed that CEBPD significantly positively regulated EGFR/PI3K pathway. Chromatin immunoprecipitation (ChIP) qPCR/Seq analysis and Luciferase reporter assay demonstrated that CEBPD binds and activates the promotor of a key ECM protein FN1 (fibronectin). In addition, the interactions of FN1 and its integrin receptors are necessary for CEBPD-induced EGFR/PI3K activation by promoting EGFR phosphorylation. Furthermore, GBM sample analysis in the database corroborated that CEBPD is positively correlated with the pathway activities of EGFR/PI3K and HIF1α, especially in highly hypoxic samples. At last, HRPs are also enriched in ECM proteins, indicating that ECM activities are important components of hypoxia induced responses in GBM. In conclusion, CEPBD plays important regulatory roles in the GBM HRG-MINW as a key TF, which activates the EGFR/PI3K pathway through ECM, especially FN1, mediated EGFR phosphorylation.

BACKGROUND: An unbalance of cell proliferation and cell apoptosis is an important mechanism in carcinogenesis, and angiogenesis also plays a crucial role in tumorigenesis. Recently, survivin has been identified as an important member of the inhibitor of apoptosis protein (IAP) family. Although it has been shown that survivin is highly expressed in gliomas, and is associated with tumorigenesis, progression, and poor prognosis of gliomas, as yet the relation of survivin expression with proliferation, apoptosis, and angiogenesis of gliomas it is still unclear.METHODS: Eighty‐three cases of brain glioma were chosen and protein expressions of survivin and proliferating cell nuclear antigen (PCNA) in glioma cells and Factor VIII‐related antigen (FVIII‐RAg) in vascular endothelial cells were investigated by immunohistochemistry. Apoptotic cells of brain glioma were screened by TdT‐mediated dUTP nick end‐labeling (TUNEL), and survivin immunoreactivity score (IRS), proliferative index (PI), apoptotic index (AI), overall daily growth (ODG), and microvessel density (MVD) in brain gliomas were measured.RESULTS: The survivin IRS, PI, AI, ODG, and MVD of brain gliomas were 3.75 ± 3.89, 28.39 ± 19.49%, 1.00 ± 0.80%, 12.19 ± 10.21%, and 62.75 ± 31.50, respectively, and all of them increased markedly with an increase in the pathologic grade of brain gliomas (P < 0.001 for all). PI, ODG, and MVD in the survivin‐positive group were significantly higher than those in the survivin‐negative group (P < 0.001 for all). PI, ODG, and MVD were positively correlated with survivin IRS (P < 0.001 for all). Although there was no significant difference between AI in the survivin‐positive group or in the survivin‐negative group (P = 0.108), AI was inversely correlated with survivin IRS (P = 0.005).CONCLUSIONS: Survivin is overexpressed in brain gliomas, which may play an important role in malignant proliferation, antiapoptosis, and angiogenesis of brain gliomas. Cancer 2005. © 2005 American Cancer Society.

Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the malignant transformation in gliomas. We hypothesized that quantitative analysis of methylated genes will provide prognostic values in malignant glioma patients. We used an immunocapturing approach followed by real-time polymerase chain reaction analysis to detect altered patterns of promoter methylation in O-6-methylguanine-DNA methyltransferase (MGMT), p16INK4a, tissue inhibitor of metalloproteinase-3 (TIMP-3), and thrombospondin 1 (THBS1). The tumor tissue and paired serum as well as cerebrospinal fluid (CSF) from 66 patients with malignant gliomas were studied. Serum and CSF from 20 age-matched noncancer individuals were used as control. Promoter hypermethylation in MGMT, p16INK4a, TIMP-3, and THBS1 was detected at high frequencies in tumor tissue, serum, and CSF. None of the control serum or CSF showed aberrant methylation. Hypermethylation in serum and CSF DNA was all accompanied with methylation in the corresponding tumor tissues with 100% specificity. Highly elevated MGMT, p16INK4a, and THBS1 methylation levels in gliomas serum were the sole independent factors predicting inferior overall survival in this cohort. For progression-free survival, hypermethylation of MGMT and THBS1 in CSF were the independent prognostic factors. Multiple gene promoter hypermethylation analysis appears to be promising as a prognostic factor in glioma and as a mini-invasive tumor marker in serum and/or CSF DNA. Evaluation of these changes may help in selecting glioma patients for optimal adjuvant treatments and modifying chemotherapy.