技术资料

Small-molecule inhibitors targeting growth factor receptors have failed to show efficacy for brain cancers,potentially due to their inability to achieve sufficient drug levels in the CNS. Targeting non-oncogene tumor co-dependencies provides an alternative approach,particularly if drugs with high brain penetration can be identified. Here we demonstrate that the highly lethal brain cancer glioblastoma (GBM) is remarkably dependent on cholesterol for survival,rendering these tumors sensitive to Liver X receptor (LXR) agonist-dependent cell death. We show that LXR-623,a clinically viable,highly brain-penetrant LXRα-partial/LXRβ-full agonist selectively kills GBM cells in an LXRβ- and cholesterol-dependent fashion,causing tumor regression and prolonged survival in mouse models. Thus,a metabolic co-dependency provides a pharmacological means to kill growth factor-activated cancers in the CNS. View Publication

Diffuse intrinsic pontine gliomas (DIPGs) have a dismal prognosis and are poorly understood brain cancers. Receptor tyrosine kinases stabilized by neuron-glial antigen 2 (NG2) protein are known to induce gliomagenesis. Here,we investigated NG2 expression in a cohort of DIPG specimens (n= 50). We demonstrate NG2 expression in the majority of DIPG specimens tested and determine that tumors harboring histone 3.3 mutation express the highest NG2 levels. We further demonstrate that microRNA 129-2 (miR129-2) is downregulated and hypermethylated in human DIPGs,resulting in the increased expression of NG2. Treatment with 5-Azacytidine,a methyltransferase inhibitor,results in NG2 downregulation in DIPG primary tumor cells in vitro. NG2 expression is altered (symmetric segregation) in mitotic human DIPG and mouse tumor cells. These mitotic cells co-express oligodendrocyte (Olig2) and astrocyte (glial fibrillary acidic protein,GFAP) markers,indicating lack of terminal differentiation. NG2 knockdown retards cellular migration in vitro,while NG2 expressing neurospheres are highly tumorigenic in vivo,resulting in rapid growth of pontine tumors. NG2 expression is targetable in vivo using miR129-2 indicating a potential avenue for therapeutic interventions. This data implicates NG2 as a molecule of interest in DIPGs especially those with H3.3 mutation. View Publication

This study investigates the electrophysiological properties and functional integration of different phenotypes of transplanted human neural precursor cells (hNPCs) in immunodeficient NSG mice. Postnatal day 2 mice received unilateral injections of 100,000 GFP+ hNPCs into the right parietal cortex. Eight weeks after transplantation,1.21% of transplanted hNPCs survived. In these hNPCs,parvalbumin (PV)-,calretinin (CR)-,somatostatin (SS)-positive inhibitory interneurons and excitatory pyramidal neurons were confirmed electrophysiologically and histologically. All GFP+ hNPCs were immunoreactive with anti-human specific nuclear protein. The proportions of PV-,CR-,and SS-positive cells among GFP+ cells were 35.5%,15.7%,and 17.1%,respectively; around 15% of GFP+ cells were identified as pyramidal neurons. Those electrophysiologically and histological identified GFP+ hNPCs were shown to fire action potentials with the appropriate firing patterns for different classes of neurons and to display spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs). The amplitude,frequency and kinetic properties of sEPSCs and sIPSCs in different types of hNPCs were comparable to host cells of the same type. In conclusion,GFP+ hNPCs produce neurons that are competent to integrate functionally into host neocortical neuronal networks. This provides promising data on the potential for hNPCs to serve as therapeutic agents in neurological diseases with abnormal neuronal circuitry such as epilepsy. View Publication

One important function of endothelial cells in glioblastoma multiforme (GBM) is to create a niche that helps promote self-renewal of cancer stem-like cells (CSLC). However,the underlying molecular mechanism for this endothelial function is not known. Since activation of NOTCH signaling has been found to be required for propagation of GBM CSLCs,we hypothesized that the GBM endothelium may provide the source of NOTCH ligands. Here,we report a corroboration of this concept with a demonstration that NOTCH ligands are expressed in endothelial cells adjacent to NESTIN and NOTCH receptor-positive cancer cells in primary GBMs. Coculturing human brain microvascular endothelial cells (hBMEC) or NOTCH ligand with GBM neurospheres promoted GBM cell growth and increased CSLC self-renewal. Notably,RNAi-mediated knockdown of NOTCH ligands in hBMECs abrogated their ability to induce CSLC self-renewal and GBM tumor growth,both in vitro and in vivo. Thus,our findings establish that NOTCH activation in GBM CSLCs is driven by juxtacrine signaling between tumor cells and their surrounding endothelial cells in the tumor microenvironment,suggesting that targeting both CSLCs and their niche may provide a novel strategy to deplete CSLCs and improve GBM treatment. View Publication

Glioblastoma multiforme (GBM) is the most common primary malignant adult brain tumor and is associated with poor survival. Recently,stem-like cell populations have been identified in numerous malignancies including GBM. To identify genes whose expression is changed with differentiation,we compared transcript profiles from a GBM oncosphere line before and after differentiation. Bioinformatic analysis of the gene expression profiles identified podocalyxin-like protein (PODXL),a protein highly expressed in human embryonic stem cells,as a potential marker of undifferentiated GBM stem-like cells. The loss of PODXL expression upon differentiation of GBM stem-like cell lines was confirmed by quantitative real-time PCR and flow cytometry. Analytical flow cytometry of numerous GBM oncosphere lines demonstrated PODXL expression in all lines examined. Knockdown studies and flow cytometric cell sorting experiments demonstrated that PODXL is involved in GBM stem-like cell proliferation and oncosphere formation. Compared to PODXL-negative cells,PODXL-positive cells had increased expression of the progenitor/stem cell markers Musashi1,SOX2,and BMI1. Finally,PODXL expression directly correlated with increasing glioma grade and was a marker for poor outcome in patients with GBM. In summary,we have demonstrated that PODXL is expressed in GBM stem-like cells and is involved in cell proliferation and oncosphere formation. Moreover,high PODXL expression correlates with increasing glioma grade and decreased overall survival in patients with GBM. View Publication

Adipose tissue-derived mesenchymal stromal cells (AT-MSCs) are good candidates for cell therapy due to the accessibility of fat tissue and the abundance of AT-MSCs therein. Neurospheres are free-floating spherical condensations of cells with neural stem/progenitor cell (NSPC) characteristics that can be derived from AT-MSCs. The aims of this study were to examine the influence of oxygen (O2) tension on generation of neurospheres from canine AT-MSCs (AT-cMSCs) and to develop a hypoxic cell culture system to enhance the survival and therapeutic benefit of generated neurospheres. AT-cMSCs were cultured under varying oxygen tensions (1%,5% and 21%) in a neurosphere culture system. Neurosphere number and area were evaluated and NSPC markers were quantified using real-time quantitative PCR (qPCR). Effects of oxygen on neurosphere expression of hypoxia inducible factor 1,α subunit (HIF1A) and its target genes,erythropoietin receptor (EPOR),chemokine (C-X-C motif) receptor 4 (CXCR4) and vascular endothelial growth factor (VEGF),were quantified by qPCR. Neural differentiation potential was evaluated in 21% O2 by cell morphology and qPCR. Neurospheres were successfully generated from AT-cMSCs at all O2 tensions. Expression of nestin mRNA (NES) was significantly increased after neurosphere culture and was significantly higher in 1% O2 compared to 5% and 21% O2. Neurospheres cultured in 1% O2 had significantly increased levels of VEGF and EPOR. There was a significant increase in CXCR4 expression in neurospheres generated at all O2 tensions. Neurosphere culture under hypoxia had no negative effect on subsequent neural differentiation. This study suggests that generation of neurospheres under hypoxia could be beneficial when considering these cells for neurological cell therapies. View Publication

Diffuse intrinsic pontine gliomas (DIPGs) represent a particularly lethal type of pediatric brain cancer with no effective therapeutic options. Our laboratory has previously reported the development of genetically engineered DIPG mouse models using the RCAS/tv-a system,including a model driven by PDGF-B,H3.3K27M,and p53 loss. These models can serve as a platform in which to test novel therapeutics prior to the initiation of human clinical trials. In this study,an in vitro high-throughput drug screen as part of the DIPG preclinical consortium using cell-lines derived from our DIPG models identified BMS-754807 as a drug of interest in DIPG. BMS-754807 is a potent and reversible small molecule multi-kinase inhibitor with many targets including IGF-1R,IR,MET,TRKA,TRKB,AURKA,AURKB. In vitro evaluation showed significant cytotoxic effects with an IC50 of 0.13 μM,significant inhibition of proliferation at a concentration of 1.5 μM,as well as inhibition of AKT activation. Interestingly,IGF-1R signaling was absent in serum-free cultures from the PDGF-B; H3.3K27M; p53 deficient model suggesting that the antitumor activity of BMS-754807 in this model is independent of IGF-1R. In vivo,systemic administration of BMS-754807 to DIPG-bearing mice did not prolong survival. Pharmacokinetic analysis demonstrated that tumor tissue drug concentrations of BMS-754807 were well below the identified IC50,suggesting that inadequate drug delivery may limit in vivo efficacy. In summary,an unbiased in vitro drug screen identified BMS-754807 as a potential therapeutic agent in DIPG,but BMS-754807 treatment in vivo by systemic delivery did not significantly prolong survival of DIPG-bearing mice. View Publication

PTEN is a tumour suppressor frequently mutated in many types of cancers. Here we show that targeted disruption of PTEN leads to neoplastic transformation of human neural stem cells (NSCs),but not mesenchymal stem cells. PTEN-deficient NSCs display neoplasm-associated metabolic and gene expression profiles and generate intracranial tumours in immunodeficient mice. PTEN is localized to the nucleus in NSCs,binds to the PAX7 promoter through association with cAMP responsive element binding protein 1 (CREB)/CREB binding protein (CBP) and inhibits PAX7 transcription. PTEN deficiency leads to the upregulation of PAX7,which in turn promotes oncogenic transformation of NSCs and instates 'aggressiveness' in human glioblastoma stem cells. In a large clinical database,we find increased PAX7 levels in PTEN-deficient glioblastoma. Furthermore,we identify that mitomycin C selectively triggers apoptosis in NSCs with PTEN deficiency. Together,we uncover a potential mechanism of how PTEN safeguards NSCs,and establish a cellular platform to identify factors involved in NSC transformation,potentially permitting personalized treatment of glioblastoma. View Publication

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor,with dismal survival outcomes. Recently,cancer stem cells (CSCs) have been demonstrated to play a role in therapeutic resistance and are considered to be the most likely cause of cancer relapse. The identification of CSCs is an important step toward finding new and effective ways to treat GBM. Tenascin-C (TNC) protein has been identified as a potential marker for CSCs in gliomas based on previous work. Here,we have investigated the expression of TNC in tissue microarrays including 17 GBMs,18 WHO grade III astrocytomas,15 WHO grade II astrocytomas,4 WHO grade I astrocytomas,and 7 normal brain tissue samples by immunohistochemical staining. TNC expression was found to be highly associated with the grade of astrocytoma. It has a high expression level in most of the grade III astrocytomas and GBMs analyzed and a very low expression in most grade II astrocytomas,whereas it is undetectable in grade I astrocytomas and normal brain tissues. Double-immunofluorescence staining for TNC and CD133 in GBM tissues revealed that there was a high overlap between theses two positive populations. The results were further confirmed by flow cytometry analysis of TNC and CD133 in GBM-derived stem-like neurospheres in vitro. A limiting dilution assay demonstrated that the sphere formation ability of CD133(+)/TNC(+) and CD133(-)/TNC(+) cell populations is much higher than that of the CD133(+)/TNC(-) and CD133(-)/TNC(-) populations. These results suggest that TNC is not only a potential prognostic marker for GBM but also a potential marker for glioma CSCs,where the TNC(+) population is identified as a CSC population overlapping with part of the CD133(-) cell population. View Publication