Purpose: BRAF is frequently activated by gene fusion or point mutation in pilocytic astrocytoma, the most common pediatric brain tumor. We investigated the functional effect of constitutive BRAF activation in normal human neural stem and progenitor cells to determine its role in tumor induction in the brain. Experimental Design: The constitutively active BRAF^V600E allele was introduced into human neurospheres, and its effects on MAPK (mitogen-activated protein kinase) signaling, proliferation, soft agarose colony formation, stem cell phenotype, and induction of cellular senescence were assayed. Immunohistochemistry was used to examine p16^INK4a levels in pilocytic astrocytoma. Results: BRAF^V600E expression initially strongly promoted colony formation but did not lead to significantly increased proliferation. BRAF^V600E-expressing cells subsequently stopped proliferating and induced markers of oncogene-induced senescence including acidic β-galactosidase, PAI-1, and p16^INK4a whereas controls did not. Onset of senescence was associated with decreased expression of neural stem cell markers including SOX2. Primary pilocytic astrocytoma cultures also showed induction of acidic β-galactosidase activity. Immunohistochemical examination of 66 pilocytic astrocytomas revealed p16^INK4a immunoreactivity in the majority of cases, but patients with tumors negative for p16^INK4a had significantly shorter overall survival. Conclusions: BRAF activation in human neural stem and progenitor cells initially promotes clonogenic growth in soft agarose, suggesting partial cellular transformation, but oncogene-induced senescence subsequently limits proliferation. Induction of senescence by BRAF may help explain the low-grade pathobiology of pilocytic astrocytoma, whereas worse clinical outcomes associated with tumors lacking p16^INK4a expression could reflect failure to induce senescence or an escape from oncogene-induced senescence. Clin Cancer Res; 17(11); 3590–9. ©2011 AACR.

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.

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.

Atypical teratoid rhabdoid tumor (AT/RT) is among the most fatal of all pediatric brain tumors. Aside from loss of function mutations in the SMARCB1 (BAF47/INI1/SNF5) chromatin remodeling gene, little is known of other molecular drivers of AT/RT. LIN28A and LIN28B are stem cell factors that regulate thousands of RNAs and are expressed in aggressive cancers. We identified high-levels of LIN28A and LIN28B in AT/RT primary tumors and cell lines, with corresponding low levels of the LIN28-regulated microRNAs of the let-7 family. Knockdown of LIN28A by lentiviral shRNA in the AT/RT cell lines CHLA-06-ATRT and BT37 inhibited growth, cell proliferation and colony formation and induced apoptosis. Suppression of LIN28A in orthotopic xenograft models led to a more than doubling of median survival compared to empty vector controls (48 vs 115 days). LIN28A knockdown led to increased expression of let-7b and let-7g microRNAs and a down-regulation of KRAS mRNA. AT/RT primary tumors expressed increased mitogen activated protein (MAP) kinase pathway activity, and the MEK inhibitor selumetinib (AZD6244) decreased AT/RT growth and increased apoptosis. These data implicate LIN28/RAS/MAP kinase as key drivers of AT/RT tumorigenesis and indicate that targeting this pathway may be a therapeutic option in this aggressive pediatric malignancy.