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In JoVE (1)
Other Publications (23)
- The Journal of Biological Chemistry
- Proceedings of the National Academy of Sciences of the United States of America
- Developmental Biology
- Journal of Neuroscience Research
- Brain Research. Developmental Brain Research
- No Shinkei Geka. Neurological Surgery
- The Journal of Cell Biology
- Pediatric Research
- Stem Cells (Dayton, Ohio)
- Journal of Neuroscience Research
- Cancer Cell
- Neuroscience Research
- Current Stem Cell Research & Therapy
- Methods in Molecular Biology (Clifton, N.J.)
- No Shinkei Geka. Neurological Surgery
- Journal of Neurosurgery
- Molecular Cancer Therapeutics
- PloS One
- World Neurosurgery
- Clinical Cancer Research : an Official Journal of the American Association for Cancer Research
Articles by Ichiro Nakano in JoVE
Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens
Kaushal Joshi1, Habibe Demir1, Ryosuke Yamada1, Takeshi Miyazaki1, Abhik Ray-Chaudhury2, Ichiro Nakano1
1Department of Neurological Surgery, The Ohio State University Medical Center, 2Department of Pathology, The Ohio State University Medical Center
Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed “tumor explant method”. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.
Other articles by Ichiro Nakano on PubMed
Stem Cell-derived Neural Stem/progenitor Cell Supporting Factor is an Autocrine/paracrine Survival Factor for Adult Neural Stem/progenitor Cells
The Journal of Biological Chemistry. Sep, 2003 | Pubmed ID: 12832409
Recent evidence suggests that adult neural stem/progenitor cells (ANSCs) secrete autocrine/paracrine factors and that these intrinsic factors are involved in the maintenance of adult neurogenesis. We identified a novel secretory molecule, stem cell-derived neural stem/progenitor cell supporting factor (SDNSF), from adult hippocampal neural stem/progenitor cells by using the signal sequence trap method. The expression of SDNSF in adult central nervous system was localized to hippocampus including dentate gyrus, where the neurogenesis persists throughout life. In induced neurogenesis status seen in ischemically treated hippocampus, the expression of SDNSF was up-regulated. As functional aspects, SDNSF protein provided a dose-dependent survival effect for ANSC following basic fibroblast growth factor 2 (FGF-2) withdrawal. ANSCs treated by SDNSF also retain self-renewal potential and multipotency in the absence of FGF-2. However, SDNSF did not have mitogenic activity, nor was it a cofactor that promoted the mitogenic effects of FGF-2. These data suggested an important role of SDNSF as an autocrine/paracrine factor in maintaining stem cell potential and lifelong neurogenesis in adult central nervous system.
Proceedings of the National Academy of Sciences of the United States of America. Dec, 2003 | Pubmed ID: 14645703
Pediatric brain tumors are significant causes of morbidity and mortality. It has been hypothesized that they derive from self-renewing multipotent neural stem cells. Here, we tested whether different pediatric brain tumors, including medulloblastomas and gliomas, contain cells with properties similar to neural stem cells. We find that tumor-derived progenitors form neurospheres that can be passaged at clonal density and are able to self-renew. Under conditions promoting differentiation, individual cells are multipotent, giving rise to both neurons and glia, in proportions that reflect the tumor of origin. Unlike normal neural stem cells, however, tumor-derived progenitors have an unusual capacity to proliferate and sometimes differentiate into abnormal cells with multiple differentiation markers. Gene expression analysis reveals that both whole tumors and tumor-derived neurospheres express many genes characteristic of neural and other stem cells, including CD133, Sox2, musashi-1, bmi-1, maternal embryonic leucine zipper kinase, and phosphoserine phosphatase, with variation from tumor to tumor. After grafting to neonatal rat brains, tumor-derived neurosphere cells migrate, produce neurons and glia, and continue to proliferate for more than 4 weeks. The results show that pediatric brain tumors contain neural stem-like cells with altered characteristics that may contribute to tumorigenesis. This finding may have important implications for treatment by means of specific targeting of stem-like cells within brain tumors.
Neural Progenitor Genes. Germinal Zone Expression and Analysis of Genetic Overlap in Stem Cell Populations
Developmental Biology. Dec, 2003 | Pubmed ID: 14651920
The identification of the genes regulating neural progenitor cell (NPC) functions is of great importance to developmental neuroscience and neural repair. Previously, we combined genetic subtraction and microarray analysis to identify genes enriched in neural progenitor cultures. Here, we apply a strategy to further stratify the neural progenitor genes. In situ hybridization demonstrates expression in the central nervous system germinal zones of 54 clones so identified, making them highly relevant for study in brain and neural progenitor development. Using microarray analysis we find 73 genes enriched in three neural stem cell (NSC)-containing populations generated under different conditions. We use the custom microarray to identify 38 "stemness" genes, with enriched expression in the three NSC conditions and present in both embryonic stem cells and hematopoietic stem cells. However, comparison of expression profiles from these stem cell populations indicates that while there is shared gene expression, the amount of genetic overlap is no more than what would be expected by chance, indicating that different stem cells have largely different gene expression patterns. Taken together, these studies identify many genes not previously associated with neural progenitor cell biology and also provide a rational scheme for stratification of microarray data for functional analysis.
Patterns of Jagged1, Jagged2, Delta-like 1 and Delta-like 3 Expression During Late Embryonic and Postnatal Brain Development Suggest Multiple Functional Roles in Progenitors and Differentiated Cells
Journal of Neuroscience Research. Feb, 2004 | Pubmed ID: 14743446
The Notch-DSL signaling system, consisting of multiple receptors and ligands, inhibits neurogenesis and promotes gliogenesis during embryonic development, but the specific function of the various ligands and receptors at later developmental stages are unknown. Here, we examined the expression pattern of four Delta, Serrate and Lag-2 (DSL) ligands, Jagged1, Jagged2, Delta-like1 (Dl1) and Delta-like 3 (Dl3), in late embryonic and postnatal rat brain by in situ hybridization. In late embryos, Jagged1, Dl1 and Dl3 mRNAs were present in the periventricular germinal epithelia, but this expression diminished during postnatal ages. Jagged1 mRNA was also expressed in the inner aspect of the dentate gyrus at early postnatal times. Dl3 was detectable in the external granule cell layer (EGL) of the cerebellum, another site of postnatal neurogenesis. Jagged2 mRNA was expressed in virtually all postnatal neurons. Jagged1 mRNA was highly expressed in several brain nuclei during postnatal development, with lower levels of expression in other grey matter regions. In white matter, Dl1 and Dl3 mRNAs were expressed during the first week of postnatal development but only the expression of Dl1 mRNA persisted through the second week. Dl1 mRNA was present at lower levels throughout grey matter during the first few weeks of development. Jagged1 mRNA was expressed in blood vessels, choroid plexus, and menninges throughout development and in the adult. Jagged2 mRNA was transiently expressed in cerebral blood vessels and choroid plexus during the first postnatal week. Taken together, these results support multiple and differing roles for the various ligands during and after central nervous system (CNS) development.
Developmental Expression of Glial Fibrillary Acidic Protein MRNA in Mouse Forebrain Germinal Zones--implications for Stem Cell Biology
Brain Research. Developmental Brain Research. Oct, 2004 | Pubmed ID: 15464225
Postnatal neural stem cells (NSCs) express the "traditional" astrocyte marker, glial fibrillary acidic protein (GFAP). Here, we analyze the ontogeny of GFAP mRNA in mouse forebrain germinal zones (GZ). On embryonic day 15, mRNA distribution is highly restricted. Subsequently, expression expands to include many cells in the GZ regions adjacent to the cortex and septum but not to the striatum. Double immunostaining for GFAP and nestin did not demonstrate extensive overlap in the GZ of adult rats, suggesting that either few of the GFAP-expressing cells are stem cells, or that nestin is not a reliable marker for stem cells in the adult rat brain. The current findings indicate that while some GFAP-expressing cells in the GZ may be NSCs, most are not likely to function in a neurogenic capacity.
No Shinkei Geka. Neurological Surgery. Aug, 2004 | Pubmed ID: 15478649
Cancers are formed by heterogeneous cell types from immature highly proliferative cells to lineage-committed differentiated cells. Transplantation studies have suggested the existence of "cancer stem cells", individual cells capable of producing an entire tumor. Recent advances in stem cell research have allowed for the demonstration of the existence of cancer stem cells in acute myeloid leukemia, breast cancer, and, most recently, in pediatric brain tumors. Each of these has some similarities with the normal stem cells in the corresponding organs. For example, leukemia stem cells express some, but not all, markers of hematopoietic stem cells. Regarding pediatric brain tumors, putative cancer stem cells were identified from medulloblastoma and also from glioma. These tumor-derived cells self-renew under clonal conditions, and differentiate into neurons and glia as well as into abnormal cells with mixed phenotypes. Interestingly, the tumor stem/progenitors, enriched in culture, maintained proliferation after 4 weeks from transplantation into neonatal rat brain. In this review, we discuss the difference as well as the similarity between tumor and normal stem cells, and also the possible clinical implication of cancer stem cells.
Maternal Embryonic Leucine Zipper Kinase (MELK) Regulates Multipotent Neural Progenitor Proliferation
The Journal of Cell Biology. Aug, 2005 | Pubmed ID: 16061694
Maternal embryonic leucine zipper kinase (MELK) was previously identified in a screen for genes enriched in neural progenitors. Here, we demonstrate expression of MELK by progenitors in developing and adult brain and that MELK serves as a marker for self-renewing multipotent neural progenitors (MNPs) in cultures derived from the developing forebrain and in transgenic mice. Overexpression of MELK enhances (whereas knockdown diminishes) the ability to generate neurospheres from MNPs, indicating a function in self-renewal. MELK down-regulation disrupts the production of neurogenic MNP from glial fibrillary acidic protein (GFAP)-positive progenitors in vitro. MELK expression in MNP is cell cycle regulated and inhibition of MELK expression down-regulates the expression of B-myb, which is shown to also mediate MNP proliferation. These findings indicate that MELK is necessary for proliferation of embryonic and postnatal MNP and suggest that it regulates the transition from GFAP-expressing progenitors to rapid amplifying progenitors in the postnatal brain.
Phenotypic and Functional Heterogeneity of GFAP-expressing Cells in Vitro: Differential Expression of LeX/CD15 by GFAP-expressing Multipotent Neural Stem Cells and Non-neurogenic Astrocytes
Glia. Feb, 2006 | Pubmed ID: 16267834
Recent findings show that the predominant multipotent neural stem cells (NSCs) isolated from postnatal and adult mouse brain express glial fibrillary acid protein (GFAP), a protein commonly associated with astrocytes, and that primary astrocyte cultures can contain GFAP-expressing cells that act as multipotent NSCs when transferred to neurogenic conditions. The relationship of GFAP-expressing NSCs to GFAP-expressing astrocytes is unclear, but has important implications. We compared the phenotype and neurogenic potential of GFAP-expressing cells derived from different CNS regions and maintained in vitro under different conditions. Multiple labeling immunohistochemistry revealed that both primary astrocyte cultures and adherent neurogenic cultures derived from postnatal or adult periventricular tissue contained subpopulations of GFAP-expressing cells that co-expressed nestin and LeX/CD15, two molecules associated with NSCs. In contrast, GFAP-expressing cells in similar cultures prepared from adult cerebral cortex did not express detectable levels of LeX/CD15, and exhibited no neurogenic potential. Fluorescence-activated cell sorting (FACS) of both primary astrocyte cultures and adherent neurogenic cultures for LeX/CD15 showed that GFAP-expressing cells competent to act as multipotent NSCs were concentrated in the LeX-positive fraction. Using neurosphere assays and a transgenic ablation strategy, we confirmed that the predominant NSCs in primary astrocyte and adherent neurogenic cultures were GFAP-expressing cells. These findings demonstrate that GFAP-expressing cells derived from postnatal and adult forebrain are heterogeneous in both molecular phenotype and neurogenic potential in vitro, and that this heterogeneity exists before exposure to neurogenic conditions. The findings provide evidence that GFAP-expressing NSCs are phenotypically and functionally distinct from non-neurogenic astrocytes.
Pediatric Research. Apr, 2006 | Pubmed ID: 16549549
Cancers are composed of heterogeneous cell populations ranging from highly proliferative immature cells to more differentiated cells of various cell lineages. Recent advances in stem cell research have allowed for the demonstration of the existence of cancer stem cells in acute myeloid leukemia, breast cancer, and, most recently, in brain tumors. Each of these has some similarities with the normal stem cells in the corresponding organs. In brain tumors, putative cancer stem cells have been identified from glioblastoma multiforme, medulloblastoma and ependymoma. These tumor-derived cells self-renew under clonal conditions, and differentiate into neuron- and glia-like cells as well as into abnormal cells with mixed phenotypes. The tumor stem cells, but not the rest of tumor cells form secondary tumors by transplantation into immunodeficient mouse brain. In this review, we discuss the cellular and molecular relationships between brain tumor stem cells and normal neural stem cells, and also the possible clinical implications of brain tumor stem cells.
Phosphoserine Phosphatase is Expressed in the Neural Stem Cell Niche and Regulates Neural Stem and Progenitor Cell Proliferation
Stem Cells (Dayton, Ohio). Aug, 2007 | Pubmed ID: 17495110
Phosphoserine phosphatase (PSP) metabolizes the conversion of l-phosphoserine to l-serine, classically known as an amino acid necessary for protein and nucleotide synthesis and more recently suggested to be involved in cell-to-cell signaling. Previously, we identified PSP as being enriched in proliferating neural progenitors and highly expressed by embryonic and hematopoietic stem cells, suggesting a general role in stem cells. Here we demonstrate that PSP is highly expressed in periventricular neural progenitors in the embryonic brain. In the adult brain, PSP expression was observed in slowly dividing or quiescent glial fibrillary acidic protein (GFAP)-positive cells and CD24-positive ependymal cells in the forebrain germinal zone adjacent to the lateral ventricle and within GFAP-positive cells of the hippocampal subgranular zone, consistent with expression in adult neural stem cells. In vitro, PSP overexpression promoted proliferation, whereas small interfering RNA-induced knockdown inhibited proliferation of neural stem cells derived from embryonic cortex and adult striatal subventricular zone. The effects of PSP knockdown were partially rescued by exogenous l-serine. These data support a role for PSP in neural stem cell proliferation and suggest that in the adult periventricular germinal zones, PSP may regulate signaling between neural stem cells and other cells within the stem cell niche. Disclosure of potential conflicts of interest is found at the end of this article.
Maternal Embryonic Leucine Zipper Kinase is a Key Regulator of the Proliferation of Malignant Brain Tumors, Including Brain Tumor Stem Cells
Journal of Neuroscience Research. Jan, 2008 | Pubmed ID: 17722061
Emerging evidence suggests that neural stem cells and brain tumors regulate their proliferation via similar pathways. In a previous study, we demonstrated that maternal embryonic leucine zipper kinase (Melk) is highly expressed in murine neural stem cells and regulates their proliferation. Here we describe how MELK expression is correlated with pathologic grade of brain tumors, and its expression levels are significantly correlated with shorter survival, particularly in younger glioblastoma patients. In normal human astrocytes, MELK is only faintly expressed, and MELK knockdown does not significantly influence their growth, whereas Ras and Akt overexpressing astrocytes have up-regulated MELK expression, and the effect of MELK knockdown is more prominent in these transformed astrocytes. In primary cultures from human glioblastoma and medulloblastoma, MELK knockdown by siRNA results in inhibition of the proliferation and survival of these tumors. Furthermore, we show that MELK siRNA dramatically inhibits proliferation and, to some extent, survival of stem cells isolated from glioblastoma in vitro. These results demonstrate a critical role for MELK in the proliferation of brain tumors, including their stem cells, and suggest that MELK may be a compelling molecular target for treatment of high-grade brain tumors.
Cancer Cell. Jan, 2008 | Pubmed ID: 18167333
Brain tumor stem cells (BTSC) bear some similarities to neural stem cells (NSC). Bone morphogenetic proteins (BMPs) have a proproliferative effect on early embryonic NSC, and a prodifferentiative effect on postnatal NSC. In this issue of Cancer Cell, Lee et al. demonstrate that BMPs have differing effects on different BTSC lines, either promoting or inhibiting an astrocytic-like differentiation program. This latter effect is the result of epigenetic silencing of the BMP receptor 1B (BMPR1B). These findings document the importance of the BMP signaling system in BTSC as well as that of taking heterogeneity into account when studying BTSC as potential targets for therapy.
Trifluoromethoxy-benzylated Ligands Improve Amyloid Detection in the Brain Using (19)F Magnetic Resonance Imaging
Neuroscience Research. Jan, 2009 | Pubmed ID: 18996420
The chemical properties of probes that improve amyloid detection by non-invasive (19)F magnetic resonance imaging (MRI) are of interest. We synthesized benzoxazole compounds with trifluoromethoxy groups, and found that these compounds displayed sharp (19)F nuclear magnetic resonance (NMR) signals in an assay buffer. However, the intensities of the (19)F NMR signals were dramatically reduced in mouse brain lysates. Our results indicate that the inhibitory effect of brain tissue on the (19)F NMR signals from these probes can be attributed to the hydrophobicity of the tissue. These results highlight the importance of using hydrophilic (19)F-MRI agents to avoid the inhibitory effects of brain tissues on (19)F NMR signals.
Current Stem Cell Research & Therapy. Dec, 2009 | Pubmed ID: 19500067
Central nervous system (CNS) tumors remain the leading cause of death among pediatric neoplasms. Although standard therapies cure many pediatric CNS tumors, the long-term cognitive and physical consequences of these therapies are devastating. Furthermore, recurrent disease carries a dismal prognosis. Although recent studies have focused on molecular mechanisms that underlie the initiation and progression of adult glioblastoma multiforme (GBM), these tumors differ phenotypically and at a molecular level from pediatric brain tumors. Recent investigations have identified a stem cell population, termed "brain tumor stem cells" (BTSC) within the heterogeneous cell populations that comprise malignant brain tumors which may be partly responsible for the resistance to current therapies. These have been identified in several pediatric tumors including medulloblastoma, ependymomas, and malignant gliomas. By exploiting molecular differences present within these heterogeneous populations of brain tumor cells, we may be able to achieve specific eradication of BTSC and long-lasting remissions, while causing less toxicity to normal tissues. In this review, we describe the issues surrounding the identification and characterization of BTSC, the molecular biology of BTSC for different pediatric brain tumors, and suggest future avenues for the development of treatments for this devastating disease.
Methods in Molecular Biology (Clifton, N.J.). 2009 | Pubmed ID: 19582420
Neural stem cells (NSC) self-renew and are multipotent, producing neurons and glia. Recent studies have shown that brain tumors (BT) contain cells that, like NSC, self-renew and are multipotent, producing the different types of cells found within the brain tumors. These brain tumor stem cells are a kind of cancer stem cell, competent to form tumors that mimic the parent tumor in experimental animals. Studies from our laboratory and others have demonstrated that brain tumor stem cells and NSC share similar mechanisms and pathways for proliferation. For example, we have identified that one of the AMPK/snf1 kinases, maternal embryonic leucine zipper kinase (MELK), is highly expressed in NSC and malignant brain tumors, as well as in brain tumor stem cell-enriched cell cultures. Analysis of transgenic MELK-reporter mice indicated that MELK is expressed in NSC in vivo, and our in vitro studies demonstrated that MELK is required for NSC self-renewal. We have also found that MELK is required for proliferation of putative BT stem cells. Utilizing our studies with MELK as an example, this chapter describes methods to culture NSC and BT stem cells, and to analyze the pathways, which regulate self-renewal of those cells.
Relationship Between the Tautomeric Structures of Curcumin Derivatives and Their Abeta-binding Activities in the Context of Therapies for Alzheimer's Disease
Biomaterials. May, 2010 | Pubmed ID: 20181392
Curcumin, which can exist in an equilibrium between keto and enol tautomers, binds to beta-amyloid (Abeta) fibrils/aggregates. The aim of this study was to assess the relationship between the tautomeric structures of curcumin derivatives and their Abeta-binding activities. Curcumin derivatives with keto-enol tautomerism showed high levels of binding to Abeta aggregates but not to Abeta monomers. The binding activity of the keto form analogue of curcumin to Abeta aggregates was found to be much weaker than that of curcumin derivatives with keto-enol tautomerism. The color of a curcumin derivative with keto-enol tautomerism, which was substituted at the C-4 position, changed from yellow to orange within 30 min of being combined with Abeta aggregates in physiological buffer. This resulted from a remarkable increase in the enol form with extended conjugation of double bonds upon binding. These findings suggest that curcumin derivatives exist predominantly in the enol form during binding to Abeta aggregates, and that the enolization of curcumin derivatives is crucial for binding to Abeta aggregates. The keto-enol tautomerism of curcumin derivatives may be a novel target for the design of amyloid-binding agents that can be used both for therapy and for amyloid detection in Alzheimer's disease.
[Cancer Stem Cells in Malignant Glioma-the Mechanism of Cancer Initiation and the Therapeutic Development]
No Shinkei Geka. Neurological Surgery. Oct, 2010 | Pubmed ID: 21041889
Malignant glioma is one of the most lethal diseases in adulthood. The median survival of patients with the Grade IV glioma, glioblastoma multiforme (GBM), is shorter than 15 months and the current first-line therapies for this devastating disease have only a palliative effect. The cancer stem cell hypothesis has recently attracted a great deal of attention, owing to the promise of a novel cellular target for the treatment of tumors including GBM. Recent studies have demonstrated that existence of cancer stem cells in brain tumors (BTSC) accounts, at least in part, for the intractability of malignant glioma. From the therapeutic standpoint, characterization of the mechanism for tumor initiation and maintenance of the "stem-like state" of BTSC is crucial. However, multiple heterogeneous subtypes of cancer stem cells have recently been identified from malignant glioma, making the idea of cancer stem cell complicated. In addition, in some cancer types (e.g. melanoma), a considerable proportion of tumor cells may possess the stem cell property, indicating cancer stem cells may not be a rare cell population in tumors, at least in some organs. Based on the extensive genetic and epigenetic characterization of tumor growth mechanisms, various molecularly-targeted therapies have already been applied for patients, demonstrating a varying degree of success in cancer treatment. A significant improvement in patient prognosis was achieved in several cancer types including leukemia and breast cancer. It is no doubt that continuous effort is required to bring hope for patients with malignant glioma. In this study, we summarize the recent findings and approaches in the cancer stem cell field, mainly focusing on malignant glioma stem cells, and also describe potential future directions in this area.
Journal of Neurosurgery. Mar, 2011 | Pubmed ID: 21054138
Neuro-oncology. Jun, 2011 | Pubmed ID: 21558073
Glioblastoma multiforme (GBM) is a devastating disease, and the current therapies have only palliative effect. Evidence is mounting to indicate that brain tumor stem cells (BTSCs) are a minority of tumor cells that are responsible for cancer initiation, propagation, and maintenance. Therapies that fail to eradicate BTSCs may ultimately lead to regrowth of residual BTSCs. However, BTSCs are relatively resistant to the current treatments. Development of novel therapeutic strategies that effectively eradicate BTSC are, therefore, essential. In a previous study, we used patient-derived GBM sphere cells (stemlike GBM cells) to enrich for BTSC and identified maternal embryonic leucine-zipper kinase (MELK) as a key regulator of survival of stemlike GBM cells in vitro. Here, we demonstrate that a thiazole antibiotic, siomycin A, potently reduced MELK expression and inhibited tumor growth in vivo. Treatment of stemlike GBM cells with siomycin A resulted in arrested self-renewal, decreased invasion, and induced apoptosis but had little effect on growth of the nonstem cells of matched tumors or normal neural stem/progenitor cells. MELK overexpression partially rescued the phenotype of siomycin A-treated stemlike GBM cells. In vivo, siomycin A pretreatment abraded the sizes of stemlike GBM cell-derived tumors in immunodeficient mice. Treatment with siomycin A of mice harboring intracranial tumors significantly prolonged their survival period compared with the control mice. Together, this study may be the first model to partially target stemlike GBM cells through a MELK-mediated pathway with siomycin A to pave the way for effective treatment of GBM.
Molecular Cancer Therapeutics. Oct, 2011 | Pubmed ID: 21859839
Glioblastoma (GBM) is among the most lethal of all cancers. GBM consist of a heterogeneous population of tumor cells among which a tumor-initiating and treatment-resistant subpopulation, here termed GBM stem cells, have been identified as primary therapeutic targets. Here, we describe a high-throughput small molecule screening approach that enables the identification and characterization of chemical compounds that are effective against GBM stem cells. The paradigm uses a tissue culture model to enrich for GBM stem cells derived from human GBM resections and combines a phenotype-based screen with gene target-specific screens for compound identification. We used 31,624 small molecules from 7 chemical libraries that we characterized and ranked based on their effect on a panel of GBM stem cell-enriched cultures and their effect on the expression of a module of genes whose expression negatively correlates with clinical outcome: MELK, ASPM, TOP2A, and FOXM1b. Of the 11 compounds meeting criteria for exerting differential effects across cell types used, 4 compounds showed selectivity by inhibiting multiple GBM stem cells-enriched cultures compared with nonenriched cultures: emetine, n-arachidonoyl dopamine, n-oleoyldopamine (OLDA), and n-palmitoyl dopamine. ChemBridge compounds #5560509 and #5256360 inhibited the expression of the 4 mitotic module genes. OLDA, emetine, and compounds #5560509 and #5256360 were chosen for more detailed study and inhibited GBM stem cells in self-renewal assays in vitro and in a xenograft model in vivo. These studies show that our screening strategy provides potential candidates and a blueprint for lead compound identification in larger scale screens or screens involving other cancer types.
PloS One. 2011 | Pubmed ID: 21915300
Identification of stem cell-like brain tumor cells (brain tumor stem-like cells; BTSC) has gained substantial attention by scientists and physicians. However, the mechanism of tumor initiation and proliferation is still poorly understood. CD44 is a cell surface protein linked to tumorigenesis in various cancers. In particular, one of its variant isoforms, CD44v6, is associated with several cancer types. To date its expression and function in BTSC is yet to be identified. Here, we demonstrate the presence and function of the variant form 6 of CD44 (CD44v6) in BTSC of a subset of glioblastoma multiforme (GBM). Patients with CD44(high) GBM exhibited significantly poorer prognoses. Among various variant forms, CD44v6 was the only isoform that was detected in BTSC and its knockdown inhibited in vitro growth of BTSC from CD44(high) GBM but not from CD44(low) GBM. In contrast, this siRNA-mediated growth inhibition was not apparent in the matched GBM sample that does not possess stem-like properties. Stimulation with a CD44v6 ligand, osteopontin (OPN), increased expression of phosphorylated AKT in CD44(high) GBM, but not in CD44(low) GBM. Lastly, in a mouse spontaneous intracranial tumor model, CD44v6 was abundantly expressed by tumor precursors, in contrast to no detectable CD44v6 expression in normal neural precursors. Furthermore, overexpression of mouse CD44v6 or OPN, but not its dominant negative form, resulted in enhanced growth of the mouse tumor stem-like cells in vitro. Collectively, these data indicate that a subset of GBM expresses high CD44 in BTSC, and its growth may depend on CD44v6/AKT pathway.
World Neurosurgery. Nov, 2011 | Pubmed ID: 22120228
Telomestatin Impairs Glioma Stem Cell Survival and Growth Through the Disruption of Telomeric G-quadruplex and Inhibition of the Proto-oncogene, C-Myb
Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Jan, 2012 | Pubmed ID: 22230766
PURPOSE: Glioma stem cells (GSCs) are a critical therapeutic target of glioblastoma multiforme (GBM). EXPERIMENTAL DESIGN: The effects of a G-quadruplex ligand, telomestatin (TMS), were evaluated using patient-derived GSCs, non-stem tumor cells (non-GSCs), and normal fetal neural precursors in vitro and in vivo. The molecular targets of TMS were determined by immunofluorescence in situ hybridization (iFISH) and cDNA microarray. The data was then validated by in vitro and in vivo functional assays, as well as by immunohistochemistry against 90 clinical samples.RESULTS: TMS impaired the maintenance of GSC stem cell-state by inducing apoptosis in vitro and in vivo. The migration potential of GSCs was also impaired by TMS treatment. In contrast, both normal neural precursors and non-GSCs were relatively resistant to TMS. Treatment of GSC-derived mouse intracranial tumors reduced tumor sizes in vivo without a noticeable cell death in normal brains. iFISH revealed both telomeric and non-telomeric DNA damage by TMS in GSCs but not in non-GSCs. cDNA microarray identified a proto-oncogene, c-Myb, as a novel molecular target of TMS in GSCs and pharmacodynamic analysis in TMS-treated tumor-bearing mouse brains demonstrated a reduction of c-Myb in tumors in vivo. Knockdown of c-Myb phenocopied TMS-treated GSCs both in vitro and in vivo, and restoring c-Myb by overexpression partially rescued the phenotype. Lastly, c-Myb expression was markedly elevated in surgical specimens of GBM compared to normal tissues. CONCLUSIONS: These data indicate that TMS potently eradicates GSCs through telomere disruption and c-Myb inhibition, and this study suggests a novel GSC-directed therapeutic strategy for GBM.