Brain Tumor Stem Cells Research Articles

Abstract 4235: Anti-proliferative effects of ZR2002, a novel combi-molecule with EGFR/DNA binary targeting properties compared to Gefitinib in glioblastoma cell lines and brain tumor stem cells

Abstract Glioblastoma multiforme (GBM) is the most aggressive form of primary brain tumor in adults with a survival of only 12-15 months. Brain tumor stem cells (BTSCs) contribute to tumor initiation, progression, chemo- and radioresistance, which accounts for tumor recurrence and treatment failure. Amplification of epidermal growth factor receptor (EGFR) is one of the most common genetic alterations associated with GBM aggressiveness. EGFR variant III (EGFRvIII) is an activating mutation that accounts for 60% of EGFR mutations in patients whose tumors show amplification of wild type EGFR. Despite the evidence that EGFR-induced pathway represents an attractive therapeutic target in GBM, gefitinib (Iressa™, ZD1839), an orally active, selective EGFR-tyrosine kinase inhibitor showed only a limited potency in clinical trials. ZR2002, a prototype of ‘combi-molecule’ capable of generating the binary EGFR/DNA targeting activity without requirement for hydrolytic cleavage has not been tested against BTSC. Given the ability of ZR2002 to concomitantly induce DNA damage and block EGFR-mediated signaling, we hypothesized that it would exert greater anti-proliferative activity than gefitinib in GBM tumor cell lines and BTSCs. To investigate our hypothesis, we used isogenic cell lines: U87EGFR wild-type (wt) and U87EGFRvIII (over-expressing EGFRvIII), U87MG (parental cell line) in addition to T98G, a GBM cell line known to express high levels of EGFR and BTSCs derived from patients newly diagnosed with GBM. BTSCs were cultured as neurospheres in stem cell media supplemented with growth factors (EGF, FGF), heparin and proliferation supplement. We examined the half maximal inhibitory concentration (IC50) of ZR2002 compared to gefitinib using MTT proliferation assay. Our results show that all cell lines tested were resistant to gefitinib when compared to ZR2002, which showed sub-micromolar potency in all tested cell lines following a short 2 h drug exposure. IC50s for T98G, U87-MG and U87-EGFRIII treated with ZR2002 were 0.019 μM, 0.048 μM and 0.073 μM, respectively. We further investigated the effect of ZR2002 in four different BTSC cell lines 48EF, OPK111, OPK164 and OPK161 and showed that the IC50s were in a similar range 0.027 μM, 0.014 μM, 0.089 μM and 0.019 μM, respectively. While GBM cell lines and BTSCs are highly resistant to gefitinib, they showed a greater sensitivity to ZR2002 (IC50 is less than 0.1 μM). These findings pave the way for developing single molecules with dual therapeutic modalities as a new strategy for GBM treatment. Additional pre-clinical studies are ongoing to evaluate the ability of ZR2002 to cross the blood brain barrier and its efficacy in an orthotopic tumor brain model. This research is funded by the Canadian Cancer Society grant #70217. Citation Format: Zeinab Sharifi, Jean-Claude Bertrand, Kevin Petrecca, Elliot Goodfellow, Bassam Abdulkarim, Siham Sabri. Anti-proliferative effects of ZR2002, a novel combi-molecule with EGFR/DNA binary targeting properties compared to Gefitinib in glioblastoma cell lines and brain tumor stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4235. doi:10.1158/1538-7445.AM2015-4235

Abstract 3298: ABL1 is required for Tie2-mediated DNA repair in brain tumor stem cells

Abstract Glioblastoma is the most aggressive primary brain tumor and, in spite of surgery and chemoradiotherapy, invariably recurs. The poor prognosis associated with this disease, with a median survival of 15 months, is largely caused by the striking radioresistance of these tumors. The development of new therapeutic strategies for patient with brain tumors requires the identification of key molecular pathways regulating their resistant phenotype. The abnormal function of tyrosine kinase receptors (TKRs) is a hallmark of malignant gliomas. We previously reported the expression of the TKR Tie2 in brain tumor stem cells (BTSCs) and in human surgical glioma specimens in relation to malignancy. In in vivo experiments, consisting of ionizing irradiation (IR) of mice bearing intracranial BTSCs-derived xenografts showed unexpected Tie2 nuclear localization. These results were confirmed by using immunofluorescence studies using confocal microscope and subcellular fractionation followed by Western blots. Of clinical interest, the presence of Tie2 in the nucleus is associated with radioresistance, as observed after mutagenesis of a newly discovered nuclear localization signal. In addition, upon IR, we detected increased levels of Tie2 natural ligand, Angiopoietin1 (Ang1). The blocking of the Ang1/Tie2 interaction, by the use of a soluble receptor, modulated the IR-mediated Tie2 nuclear translocation, indicating Tie2 intracellular trafficking was ligand dependent. Additionally we also found that after IR treatment, Tie2 localized in the DNA-repair foci and complexed with the H2AX, the key DNA repair protein. The data presented here clearly suggested a role of Tie2 in the DNA damage repair machinery. To test our hypothesis, we used a fluorescent reporter construct in which a functional GFP gene was reconstituted following a non-homologous end joining (NHEJ) event (gift from Dr. Gorbunova, University of Rochester), and we observed that Tie2-expressing cells displayed a more efficient NHEJ repair than Tie2 negative counterparts. Based on the recently reported role of ABL1 (cAbl) in the ATM and KAT5 mediated DNA damage repair, we explored the relationship between ABL1 and the Tie2-mediated radioresistance. Our data clearly showed that DNA repair efficiency significantly and specifically decreased by using ABL1 inhibitor but not by knocking down ABL2 expression. We further analyzed the interactions between Tie2 and chromatin and, interestingly, observed that Tie2 complexes with core histones. Collectively, our results should propel the development of preclinical studies on the combination of nuclear Tie2-targeting strategies with radiotherapy for patients with glioblastomas. Citation Format: Mohammad B. Hossain, Rehnuma Shifat, David G. Johnson, Mark T. Bedford, Mien-Chie Hung, Nahir Cortes-Santiago, Konrad Gabrusiewicz, Joy Gumin, Ravesanker Ezhilarasan, Erik P. Sulman, Frederick Lang, Raymond Sawaya, W.K. Alfred Yung, Juan Fueyo, Candelaria Gomez-Manzano. ABL1 is required for Tie2-mediated DNA repair in brain tumor stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3298. doi:10.1158/1538-7445.AM2015-3298

Abstract 3341: TP508 sensitizes cancer stem cells to radiation and delays brain cancer cell relapse in vitro

Abstract In 2014, approximately 70,000 adults and 4,500 children are expected to be diagnosed with brain cancer in the U.S alone, making this disease the second leading cause of cancer related deaths in children and young adults. Currently available therapies such as radio/chemotherapy are ineffective in completely eradicating the tumor bulk and cancer stem cells within the tumor, thus allowing recurrence of the disease. Therefore, it is important to develop novel therapeutics that sensitize cancer cells to make them more susceptible to radiation therapy and reduce the number of viable cancer stem cells. Preliminary data showed that injection of TP508, a 23 amino acid thrombin derived peptide drug, increased brain tumor shrinkage post-radiation therapy (RT) in mice. To study this phenomenon, we chose to use an in vitro model of tumor relapse that selects for and allows quantification of tumor stem cells in 3D spheroid cultures. Our goal was to determine whether TP508 could specifically sensitize cancer stem cells to the effects of RT and prevent or delay stem cell mediated in vitro tumor relapse. Medulloblastoma (Daoy) cells were grown as 3D spheroids in vitro to select for the growth of stem cells. Primary spheroids were treated on day 5 with either saline or TP508 (0.5mg/mL), and exposed to radiation (10Gy) on day 7 using a Cs137 source. One day post-RT, cells were dissociated and cultured as secondary spheroids to determine the stemness or relapse potential of the cells following RT. Daily images were taken to quantify neurosphere formation. Spheroids were also analyzed for viability, apoptosis, proliferation (PCNA), and for stem cell markers (CD133/LGR5/CD44/DCLK1/Sox-2), using western blots, flow cytometry, and immuno-fluorescent staining. TP508 treatment of Daoy cell spheroids prior to RT significantly delayed the formation of secondary spheroids, decreased cell viability, number of proliferating cells, and the number of cells expressing cancer stem cell markers. Thus, in this in vitro assay, TP508 sensitized cancer stem cells to the effects of RT and delayed relapse of brain tumor stem cells in vitro. These findings suggest that injection of TP508 prior to RT treatment may sensitize cancer stem cells to radiation and help prevent brain tumor relapse. Studies are underway to determine the mechanism by which TP508 sensitizes cancer stem cells to RT and whether TP508 has similar effects on other types of cancer stem cells. This work was supported by NCI Contract # HHSN261201300076C to DC. Citation Format: Stephanie M. Moya, Carla Kantara, Laurie Sower, Darrell H. Carney. TP508 sensitizes cancer stem cells to radiation and delays brain cancer cell relapse in vitro. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3341. doi:10.1158/1538-7445.AM2015-3341

Glioma stem cells and the occurrence of tumor blood vessels

Epithelial glioma is the most common brain cancer, accounting for 35.26%-60.69% of intracranial tumors with an average of 44.69%, and it remains the greatest challenge in the field of neurosurgery. The median survival time of patients with advanced glioma is only 12 to 18 months due to the characteristics of high aggression, and the therapeutic effect was poor though surgery, chemotherapy, and targeted drug therapy being treated. Because of the presence of heterogeneity and the differentiation disorder, only a small number of glioma cells are the source of tumor growth and metastasis, which are highly resistant to traditional treatments. They are deemed as the “seed” tumor cells as they could get rid of the effect of the treatment and reconstruct the organization of tumor. They are also termed as brain tumor stem cell (BTSC) or glioma stem cells (GSCs) since neural stem cells share similar features with them. Recent data reveal that they are directly related with invasion, angiogenesis, tolerance, chemotherapy, recurrence of glioma. Based on the research result by the team, the paper elaborates the characteristics of GSCs and the relationship with the tumor angiogenesis.

Novel association between microglia and stem cells in human gliomas: A contributor to tumour proliferation?

Brain tumour stem cells and microglia both promote the growth of astrocytomas, the commonest form of primary brain tumour, with recent emerging evidence that these cell types may interact in glioma models. It is unclear whether microglia and stem cells are associated in human gliomas. To investigate this question, we used the technique of tissue microarrays to perform a correlative study of a large number of tumour samples. We quantified immunostaining of human astrocytic tumour tissue microarrays (86 patients; World Health Organisation grade II–IV) for microglia Ionized calcium binding adaptor molecule 1 (Iba1) and CD68, and stem cell nestin, SOX2 and CD133. Ki67 was used to assess proliferation and GFAP for astrocytic differentiation. Immunoreactivity for both microglial markers and stem cell markers nestin and SOX2 significantly increased with increasing tumour grade. GFAP was higher in low grade astrocytomas. There was a positive correlation between: (i) both microglial markers and nestin and CD133, (ii) nestin and tumour cell proliferation Ki67 and (iii) both microglial markers and Ki67. SOX2 was not associated with microglia or tumour proliferation. To test the clinical relevance, we investigated the putative association of these markers with clinical outcomes. High expression for nestin and Iba1 correlated with significantly shorter survival times, and high expression for nestin, Iba1, CD68 and Ki67 was associated with faster tumour progression on univariate analysis. On multivariate analysis, nestin, CD133 and Ki67 remained significant predictors of poorer survival, after adjustment for other markers. These results confirm previous in vitro findings, demonstrating their functional relevance as a therapeutic target in humans. This is the first report of a novel correlation between microglia and stem cells that may drive human astrocytic tumour development.

Differential Targeting of Stem Cells and Differentiated Glioblastomas by NK Cells.

We have recently shown that Natural Killer (NK) cells control survival and differentiation of Cancer Stem-like Cells (CSCs) through two distinct phenotypes of cytotoxic and anergic NK cells, respectively. In this report, brain CSCs and their serum and NK cell differentiated counterparts were studied. Serum-differentiated brain CSCs were significantly less susceptible to NK cells and CTL direct cytotoxicity as well as NK cell mediated Antibody Dependent Cellular Cytotoxicity (ADCC), whereas their CSCs were highly susceptible. The levels of CD44 and EGFR were higher in brain tumor CSCs when compared to the serum-differentiated tumors. No differences could be observed for the expression of MHC class I between brain tumor stem cells and their serum-differentiated counterparts. Moreover, supernatants from the combination of IL-2 and anti-CD16mAb treated NK cells (anergized NK cells) induced resistance of brain tumor CSCs to NK cell mediated cytotoxicity. Unlike serum-differentiated CSCs, NK supernatant induced differentiation and resistance to cytotoxicity in brain CSCs correlated with the increased expression of CD54 and MHC class I. The addition of anti-MHC class I antibody moderately inhibited NK mediated cytotoxicity against untreated or serum-differentiated CSCs, whereas it increased cytotoxicity against NK supernatant differentiated tumors. Therefore, two distinct mechanisms govern serum and NK supernatant mediated differentiation of brain tumors.

Glioblastoma and calcium signaling--analysis of calcium toolbox expression.

The characteristics of a cellular calcium signal (calcium signature) are determined, at least partly, by the expression of a subset of genes encoding proteins involved in calcium entry, calcium uptake and calcium modulation. Our aim in the present work was to characterize the set of genes involved in calcium signal generation that are differentially expressed in normal brain tissues versus brain tumor and/or glioma stem cells. Public datasets were analyzed according to a four step methodology consisting of: 1. detecting the outliers by using principal component analysis of the whole transcriptome; 2. building a calcium toolbox composed of 260 genes involved in the generation and modulation of the calcium signal; 3. analyzing the calcium toolbox transcriptome of different human brain areas and 4. detecting genes from the calcium toolbox preferentially expressed in tumor tissues or tumor cells compared to normal brain tissues. Our approach was validated on normal brain tissue. Tumor sample analysis allowed us to disclose a set of eighteen genes characteristic of glioblastoma tissues or glioma stem cells. Interpreting the set of genes highlighted in the study led us to propose that i) the mechanism of store operated calcium entry is strongly perturbed in cancer cells and tissues, ii) the process of calcium reuptake into mitochondria is more important in cancer cells and tissues than in their normal counterparts and iii) these two mechanisms may be coupled in at least one subgroup of the glioblastoma stem cells.

Progression of brain tumor stem cell markers

Brain tumor stem cells （BTSCs） play a key role in tumorigenesis and tumor development.There are a group of special markers in the surface of brain tumor stem cells,which can sorts stem cells,precursor cells and cancer stem cells.In recent years,the studies of CD133,Nestin,high-mobility group A1,A2B5 have made some breakthroughs. Key words: Neoplastic stem cells; Glioma

Deregulated proliferation and differentiation in brain tumors.

Neurogenesis, the generation of new neurons, is deregulated in neural stem cell (NSC)- and progenitor-derived murine models of malignant medulloblastoma and glioma, the most common brain tumors of children and adults, respectively. Molecular characterization of human malignant brain tumors, and in particular brain tumor stem cells (BTSCs), has identified neurodevelopmental transcription factors, microRNAs, and epigenetic factors known to inhibit neuronal and glial differentiation. We are starting to understand how these factors are regulated by the major oncogenic drivers in malignant brain tumors. In this review, we will focus on the molecular switches that block normal neuronal differentiation and induce brain tumor formation. Genetic or pharmacological manipulation of these switches in BTSCs has been shown to restore the ability of tumor cells to differentiate. We will discuss potential brain tumor therapies that will promote differentiation in order to reduce treatment resistance, suppress tumor growth, and prevent recurrence in patients.

SC-27 * CD13 REPRESENTS A NOVEL BRAIN TUMOR STEM CELL MARKER FOR GLIOBLASTOMA

Glioblastoma multiforme (GBM) is a very deadly and highly vascularized tumor, but targeting glioma angiogenesis by vascular endothelial growth factor inhibition has been minimally successful. Thus, understanding the molecular mechanisms that control glioma angiogenesis and progression could lead to significant new therapies. We previously described that mitochondrial DNA (mtDNA) depletion induces increased tumor angiogenesis, decreased mouse survival, increased resistance to chemotherapy, and an increase in CD133 (a well-known stem cell marker). Because glioma enriched with brain tumor stem cells (BTSCs) share similar properties with mtDNA depleted glioma cells, we hypothesize that genes up-regulated in mitochondrial depleted cells could be signature markers of BTSCs. Thus, mtDNA could be essential for the maintenance and expansion of BTSC populations. We ran an RT Profiler PCR array for angiogenesis on both U251 ρ° cells depleted of mtDNA and on the parental, isogenic U251 glioma cells. CD13 mRNA expression was increased 200 fold in ρ° cells over U251 cells. Using fluorescence-activated cell sorting and immunocytochemisty we found that CD13 is significantly increased in ρ° cells over U251 cells, and is not regulated by hypoxia (1% O2). Purification of the CD13+ population by magnetic-activated cell sorting revealed that these cells form neurospheres when cultured in Neurobasal (NB) medium. In addition, 60% of CD13+ cells were also CD133+ in NB medium, while only 10% of CD13+ cells were also CD133+ in DMEM/F-12 medium. These findings suggest CD13 may be a marker of cancer stem cells in GBM. Further elucidation of the molecular mechanisms controlling the expression of CD13 may reveal novel therapuetic targets capable of improving the treatment of GBM.

ME-05 * COUNTERATTACKING THE FORCE BEHIND GLIOMA INVASION

Brain tumor dispersal is the major cause behind the extreme difficulty of treatment and the inevitable recurrence of malignant gliomas. Determining the mechanism underlying the invasion of brain tumor cells will suggest rational therapeutic strategies. Importantly, acquisition of a highly aggressive invasive phenotype is being observed in patients with recurrent malignant gliomas treated with antiangiogenesis therapy and is considered to be responsible for the therapy failure. Although the cause for this tumor behavior remains elusive, it has been hypothesized that the tumor microenvironment may be one major influence in the invasive development. In this regard, we have recently described the recruitment of Tie2-expressing monocytes (TEMs) into the tumor/normal brain interface upon anti-VEGF therapies in preclinical models and in human surgical specimens, and reported that this specific myeloid population is responsible for the heightened invasive properties of brain tumor stem cells. Here we investigated changes in the cytokine milieu of these tumors and identified increased expression of Angiopoietin 2 (Ang2), a Tie2 ligand, after therapies targeting either VEGF (bevacizumab, aflibercept) or VEGFR (DC101), but no after standard chemotherapy. In vitro and in vivo models were combined to decipher the role of Ang2, and we observed that expression of Ang2 functioned as a potent chemoattractant of TEMs. Moreover, we observed that Ang2 stimulates the tissue remodeling properties of TEMs, further enhancing the pro-invasive properties of this tumor recruited population. Of interest, modulation of molecules blocking the Ang/Tie2 axes in a preclinical model jeopardizes the tumor recruitment of TEMs and drastically resulted in a complete abolition of the heightened invasion induced by antiangiogenesis therapy. These results might have important implications in the clinical scenario since decrease of the invasive tumor pattern could potentially render recurrent tumors amenable to surgical removal ultimately prolonging the survival of patients with malignant gliomas.

BI-02 * FUNCTIONAL CHARACTERIZATION OF NOVEL BIOMARKERS IN SELECTING FOR SUBTYPE SPECIFIC MEDULLOBLASTOMA PHENOTYPES

Major research efforts have focused on the isolation and characterization of brain tumor stem cells, or propagating cells (BTPC) in a variety of brain cancers. Elucidating cell surface marker profiles that can be used to selectively isolate this cellular population is an imperative first step in the development of targeted therapies. Medulloblastoma (MB) is the most common form of pediatric brain cancer. MB is divided into 4 molecular subgroups; Wnt, Sonic Hedgehog (SHH), Group 3 and Group 4. Given the variable results obtained for currently utilized markers, as well as the cellular heterogeneity within and between MB sub-groups, it is likely there are additional surface marker profiles capable of selecting for sub-type specific MB BTPCs. We set out to identify novel surface marker combinations capable of selecting for TPCs in SHH MB. We employed the new BD Bioscience Lyoplate screening platform to compare 242 human cell surface marker levels across high and low self-renewing SHH MB sub-clones. The top 25 markers were refined by evaluating expression levels in Shh vs Group 3,4 and Wnt variants in transcriptome datasets representing 548 patient samples. Four markers, CD271, CD106/VCAM1, EGFR and CD171/NCAM-L1 showed consistent differential expression in the SHH subtype relative to the other variants. Flow cytometry validation in additional cell lines confirmed these findings. As a proof of principle, functional characterization of CD271 in SHH MB in vitro and in vivo was performed. Using fluorescence activated cell sorting and gain/loss of function studies, our results suggest that CD271 selects for MB progenitor cells. This work highlights a new approach to screening for differentially expressed surface markers across matched samples. We delineated a cell surface fingerprint for BTPC populations from MB molecular variants, however the utility can be seen in normal stem cell biology and across all forms of cancer.

Abstract 3944: Caveolin-mediated Tie2 nuclear translocation results in enhanced NHEJ repair and glioma radioresistance

Abstract Glioblastoma is the most frequent subtype of primary brain tumor in adults and are resistant to current strategies of surgery, irradiation, and chemotherapy. Patients suffering from this disease exhibit a median survival that ranges from 9 to 15 months. Glioblastomas invariably recur after therapy due to the presence of cells exhibiting a multitherapy-resistance phenotype. To decipher the aberrant DNA repair pathways that enable tumor cells to survive DNA damage upon chemotherapy and radiotherapy should guide us to provide our patients with a more efficacious therapy. The abnormal function of tyrosine kinase receptors (TKRs) is a hallmark of malignant gliomas. Our group reported the expression of the TKR Tie2 in in brain tumor stem cells (BTSCs) and in human surgical glioma specimens in relation to malignancy. In in vivo experiments, consisting of ionizing irradiation (IR) of mice bearing intracranial BTSCs-derived xenografts showed unexpected Tie2 nuclear localization. We observed that Tie2 traffics from the cellular membrane to the nucleus upon IR stimuli, as assessed by immunoflurorescence studies using confocal microscope and subcellular fractionation followed by Western blots. This phenomenon is ligand dependent, as increased levels of Tie2 natural ligand, Angiopoietin1 (Ang1), were observed after ionizing radiation, and nuclear Tie2 levels were decreased using soluble Tie2, which blocks the Ang1/Tie2 interaction. Of clinical significance, the presence of Tie2 in the nucleus is significantly associated with radioresistance, as observed by cell viability and clonogenic assays. Tie2 trafficking was associated to Caveolin-1, and this functional complex was disturbed when Caveolin-1 inhibitor or siCaveolin-1 were used, resulting in decreased Tie2 nuclear levels. In addition, we observed that upon IR treatment, nuclear Tie2 bound to DNA/protein complexes, and specifically to the key DNA repair protein γH2AX, as a component of the DNA-repair foci. Based on these results, we hypothesized that Tie2 was involved in DNA damage repair and focused on one of the main pathways involved in double strand break (IR induced), the non-homologous end-joining (NHEJ) repair mechanism. By using a fluorescent reporter construct in which a functional GFP gene is reconstituted following an NHEJ event (gift from Dr. Gorbunova, University of Rochester), we observed that Tie2-expressing cells displayed a more efficient NHEJ repair than Tie2 negative counterparts. Mutation of a Tie2 nuclear localization signal significantly decreased NHEJ efficiency repair and radiosensitization of BTSCs, suggesting a role of nuclear Tie2 in DNA damage repair, specifically in the NHEJ repair mechanism. Collectively, our results should propel the development of preclinical studies of the combination of nuclear Tie2-targeting strategies with radiotherapy for patients with glioblastomas. Citation Format: Mohammad B. Hossain, Nahir Cortes-Santiago, Xuejun Fan, Konrad Gabrusiewicz, Joy Gumin, Erik P. Sulman, Frederick Lang, Raymond Sawaya, W.K.Alfred Yung, Juan Fueyo, Candelaria Gomez-Manzano. Caveolin-mediated Tie2 nuclear translocation results in enhanced NHEJ repair and glioma radioresistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3944. doi:10.1158/1538-7445.AM2014-3944

Microenvironment and brain tumor stem cell maintenance: impact of the niche.

There is a lot of experimental evidence that brain tumors might be sustained by a subpopulation of immature cells, so-called brain tumor stem cells (BTSCs), which do not only drive tumor formation but are highly resistant to conventional therapies. Recent findings suggest a critical role of the molecular and cellular tumor microenvironment in which these cells reside for the maintenance of stem cell properties and therapy resistance. However, detection of different BTSC phenotypes even in the same patient tumor and the observation of a marked plasticity due to instability of the BTSC phenotype caused by the environmental niche have led to a controversial discussion on the validity of the cancer stem cell concept. What complicates the situation even more is that there are different types of niches and little is known about the interplay of the niche components with one another and with different types of BTSCs in the context of stem cell maintenance. In this article we review our current knowledge on different BTSC phenotypes and the cellular components and physiology of the niche in which these cells reside. In addition, we will summarize the molecular and functional interaction of niche cells and niche conditions and how this impacts on BTSC maintenance.

Targeting Self-Renewal in High-Grade Brain Tumors Leads to Loss of Brain Tumor Stem Cells and Prolonged Survival

Cancer stem cells (CSCs) have been suggested as potential therapeutic targets for treating malignant tumors, but the in vivo supporting evidence is still missing. Using a GFP reporter driven by the promoter of the nuclear receptor tailless (Tlx), we demonstrate that Tlx(+) cells in primary brain tumors are mostly quiescent. Lineage tracing demonstrates that single Tlx(+) cells can self-renew and generate Tlx(-) tumor cells in primary tumors, suggesting that they are brain tumor stem cells (BTSCs). After introducing a BTSC-specific knock-out of the Tlx gene in primary mouse tumors, we observed a loss of self-renewal of BTSCs and prolongation of animal survival, accompanied by induction of essential signaling pathways mediating cell-cycle arrest, cell death, and neural differentiation. Our study demonstrates the feasibility of targeting glioblastomas and indicates the suitability of BTSCs as therapeutic targets, thereby supporting the CSC hypothesis.

Targeting c-Met receptor overcomes TRAIL-resistance in brain tumors.

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) induced apoptosis specifically in tumor cells. However, with approximately half of all known tumor lines being resistant to TRAIL, the identification of TRAIL sensitizers and their mechanism of action become critical to broadly use TRAIL as a therapeutic agent. In this study, we explored whether c-Met protein contributes to TRAIL sensitivity. We found a direct correlation between the c-Met expression level and TRAIL resistance. We show that the knock down c-Met protein, but not inhibition, sensitized brain tumor cells to TRAIL-mediated apoptosis by interrupting the interaction between c-Met and TRAIL cognate death receptor (DR) 5. This interruption greatly induces the formation of death-inducing signaling complex (DISC) and subsequent downstream apoptosis signaling. Using intracranially implanted brain tumor cells and stem cell (SC) lines engineered with different combinations of fluorescent and bioluminescent proteins, we show that SC expressing a potent and secretable TRAIL (S-TRAIL) have a significant anti-tumor effect in mice bearing c-Met knock down of TRAIL-resistant brain tumors. To our best knowledge, this is the first study that demonstrates c-Met contributes to TRAIL sensitivity of brain tumor cells and has implications for developing effective therapies for brain tumor patients.

Downregulation of VEGF expression attenuates malignant biological behavior of C6 glioma stem cells

Several lines of direct evidence show that gliomas express high levels of vascular endothelial growth factor (VEGF). VEGF can promote the growth of gliomas through angiogenesis. It is believed that gliomas originate in the brain tumor stem cells (BTSCs). However, the direct effect of VEGF on the biological behavior of BTSCs has not been completely elucidated. In this study, we established C6 glioma stem cells (C6GSCs) from the C6 glioma cells. Furthermore, we suppressed the VEGF expression of C6GSCs using lentiviral vector-VEGF shRNA. After transfection, the VEGF expression of C6GSCs was downregulated significantly. The proliferation and invasion capacity of transfected C6GSCs was impaired and the ability of differentiation was enhanced. The data demonstrate that downregulation of VEGF expression attenuates malignant biological behavior of C6GSCs. RNA interference of VEGF expression implies an effective anti-gliomas strategy.

BuGZ Is Required for Bub3 Stability, Bub1 Kinetochore Function, and Chromosome Alignment

During mitosis, the spindle assembly checkpoint (SAC) monitors the attachment of kinetochores (KTs) to the plus ends of spindle microtubules (MTs) and prevents anaphase onset until chromosomes are aligned and KTs are under proper tension. Here, we identify a SAC component, BuGZ/ZNF207, from an RNAi viability screen in human glioblastoma multiforme (GBM) brain tumor stem cells. BuGZ binds to and stabilizes Bub3 during interphase and mitosis through a highly conserved GLE2p-binding sequence (GLEBS) domain. Inhibition of BuGZ results in loss of both Bub3 and its binding partner Bub1 from KTs, reduction of Bub1-dependent phosphorylation of centromeric histone H2A, attenuation of KT-based Aurora B kinase activity, and lethal chromosome congression defects in cancer cells. Phylogenetic analysis indicates that BuGZ orthologsarehighly conserved among eukaryotes, but are conspicuously absent from budding and fission yeasts. These findings suggest that BuGZ has evolved to facilitate Bub3 activity and chromosome congression in higher eukaryotes.

2P180 量子ドットを用いた脳がん幹細胞表面の膜タンパク質の運動解析(12. 細胞生物的課題,ポスター,第52回日本生物物理学会年会(2014年度))

2P180 量子ドットを用いた脳がん幹細胞表面の膜タンパク質の運動解析(12. 細胞生物的課題,ポスター,第52回日本生物物理学会年会(2014年度))

Lactate dehydrogenase A silencing in IDH mutant gliomas

Mutations of the isocitrate dehydrogenase 1 and 2 gene (IDH1/2) were initially thought to enhance cancer cell survival and proliferation by promoting the Warburg effect. However, recent experimental data have shown that production of 2-hydroxyglutarate by IDH mutant cells promotes hypoxia-inducible factor (HIF)1α degradation and, by doing so, may have unexpected metabolic effects. We used human glioma tissues and derived brain tumor stem cells (BTSCs) to study the expression of HIF1α target genes in IDH mutant ((mt)) and IDH wild-type ((wt)) tumors. Focusing thereafter on the major glycolytic enzyme, lactate dehydrogenase A (LDHA), we used standard molecular methods and pyrosequencing-based DNA methylation analysis to identify mechanisms by which LDHA expression was regulated in human gliomas. We found that HIF1α-responsive genes, including many essential for glycolysis (SLC2A1, PDK1, LDHA, SLC16A3), were underexpressed in IDH(mt) gliomas and/or derived BTSCs. We then demonstrated that LDHA was silenced in IDH(mt) derived BTSCs, including those that did not retain the mutant IDH1 allele (mIDH(wt)), matched BTSC xenografts, and parental glioma tissues. Silencing of LDHA was associated with increased methylation of the LDHA promoter, as was ectopic expression of mutant IDH1 in immortalized human astrocytes. Furthermore, in a search of The Cancer Genome Atlas, we found low expression and high methylation of LDHA in IDH(mt) glioblastomas. To our knowledge, this is the first demonstration of downregulation of LDHA in cancer. Although unexpected findings, silencing of LDHA and downregulation of several other glycolysis essential genes raise the intriguing possibility that IDH(mt) gliomas have limited glycolytic capacity, which may contribute to their slow growth and better prognosis.