Glioblastoma Cancer Stem Cells Research Articles

Abstract 427: Sox2 modulation of cancer stem cell behavior in GBM

Abstract Background and Objective: Glioblastoma (GBM) is the most aggressive primary brain tumor. GBM cells are phenotypically plastic and can present cancer stem cell (CSC) properties, such as the ability to self-renew and differentiate into the cells comprising the bulk of the tumor, phenocopying the parental tumor in mouse xenografts. GBM CSCs express neural stem cell (NSC) markers, including Sox2, a HMG-box transcription factor that regulates the expression of genes associated with self-renewal and differentiation. Culturing dissociated GBM tumors in serum-containing medium typically results in loss of the CSC phenotype and downregulation of Sox2, while serum-free neurosphere medium (NM) is used for selection and propagation of CSCs. We have previously identified a GBM specimen (GS1) which, contrary to the norm, retained Sox2 expression in low passage serum culture, along with the ability to dedifferentiate into neurospheres in vitro, and to form tumors in rodents (deCarvalho et al., Stem Cells 2010, 28:181-190). Here we investigate the endogenous expression of Sox2 in GBM biopsies, xenograft tumors, and cultured cells, and test the hypothesis that Sox2 may be a driver of the cancer stem cell phenotype in GBMs. Experimental Approaches: Endogenous expression of Sox2, downstream targets, and cell lineage markers, was determined by immunohistochemistry. miRNAs targeting Sox2 were stably expressed in GBM cells. The effect of Sox2 knockdown on clonogenicity, global gene expression, proliferation in vitro, tumor formation and morphology was determined. Results: Sox2 expression was heterogeneous within and among the 26 GBM surgical specimens tested, ranging from 1% to over 80% positive nuclei. Robust Sox2 expression was observed in over 80% of 56 GBM orthotopic xenografts analyzed. Sox2 positive cells co-expressed mesenchymal (vimentin), neural (nestin), and astrocytic (GFAP) markers. Knocking down Sox2 expression significantly impaired the ability of GS1 cells to form neurospheres, without affecting cell proliferation, significantly altered the expression of known target genes, such as Nestin, and genes in various signaling pathways, and affected xenograft tumor formation. Sox2 protein levels in GBMs were further regulated by post-transcriptional mechanisms. Conclusions: Sox2 is part of the signature gene set for the proneural GBM subclass. Our results show that Sox2 protein expression is ubiquitous in GBM tumors and xenografts, independent of patient survival or treatment status. Sox2 is a master regulator of embryonic and neural stem cell phenotype, and our data contributes to the view that it may have an important role in the plasticity of GBM cells in acquiring CSC phenotype, with possible implications for the pathology of GBMs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 427. doi:1538-7445.AM2012-427

Abstract 3371: Targeting glioblastoma stem cell pathways through phage display screening

Abstract Glioblastomas are the most common and lethal primary brain tumor with conventional therapy offering only palliation. Their resistance to radiotherapy and chemotherapy has been hypothesized to be due in part to the therapeutic resistance of cancer stem cells (CSCs), a subpopulation of cancer cells functionally defined by the ability to self renew and propagate tumors. Development of effective and safe anti-CSC therapeutics will require a selective targeting of CSCs without disruption of normal stem cell maintenance. As phage display technology has been used in cancer to identify, target, and image tumor cells, we employed phage display technology to identify novel proteins specific to the functioning of brain tumor stem cells. A M13 bacteriophage peptide library consisting of 109 different combinations of 7 amino acid long peptide sequences was screened for binding to functionally validated CSCs in both in vitro and in vivo settings. In vitro screening was performed against CSCs isolated from human glioblastoma tumors grown in culture following negative selection on non-CSCs. In parallel, in vivo screening was also performed by injecting the random phage library into immunocompromised mice with either subcutaneous or intracranially xenografted glioblastoma cell lines. The xenografted tumors were resected and divided into stem and non-stem cells prospectively. Phage peptides bound to CSCs were collected in order to isolate peptide sequences that selectively target the CSC subpopulation. In silico analysis of the recovered peptide sequences from both screens using a protein BLAST search revealed several novel proteins that may play an important role in the maintenance and functioning of glioblastoma CSCs. Gene expression analysis showed that several of these genes are preferentially upregulated in glioma CSCs when compared to their non-stem counterparts, suggesting these genes may play a vital role in the unique functions of CSCs and be used to target and image CSCs. These studies validate phage display screening as a method to discover novel proteins and pathways that permit interrogation of CSCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3371. doi:1538-7445.AM2012-3371

The RNA‐binding protein Imp2 regulates oxidative phosporylation that is key to glioblastoma cancer stem cell maintenance

Insulin‐like growth factor 2 mRNA binding protein 2 (Imp2, IGF2BP2) is expressed during embryonic brain development and associated with several types of cancer. We have found that 75% cases of glioblastoma multiforme (GBM), the most malignant type of glioma, express Imp2, whereas low grade gliomas and normal adult brain do not. Interestingly expression of Imp2 was particularly elevated in GBM cancer stem cells (CSC). Immunoprecipitation of Imp2‐containing ribonucleoprotein complexes from CSC spheroids revealed that mRNAs as well as proteins bound by Imp2, including the respiratory complex I protein NDUFS3, play a role in mitochondrial energy production. Imp2 depletion severely reduced GBM CSC ability to form spheroids and to initiate tumor growth. Proliferation, ATP content and oxygen consumption rate were also diminished in GBM CSC depleted of Imp2 due to an Imp2 depletion‐dependent defect in complex I assembly and activity. Inhibition of oxidative phosphorylation (OxPhos) in GBM CSC with the selective respiratory complex I inhibitor rotenone, mimicked the effect of Imp2 silencing. By contrast, GBM CSC‐derived non tumorigenic cells were not affected by rotenone, but were sensitive to the anaerobic glycolysis inhibitor oxamic acid. Our observations provide evidence for an unsuspected function of Imp2 in the control of OxPhos and demonstrate its key role in GBM CSC maintenance.

CD90 is Identified as a Marker for Cancer Stem Cells in High-Grade Gliomas Using Tissue Microarrays

CD90 is Identified as a Marker for Cancer Stem Cells in High-Grade Gliomas Using Tissue Microarrays

BMX and Its Role in Inflammation, Cardiovascular Disease, and Cancer

Bone marrow kinase on chromosome X (BMX) is a cytosolic tyrosine kinase and a member of the TEC kinase family. BMX is expressed in hematopoietic cells of the myeloid lineage where it participates in the immune response. It is also involved in the response to ischemia and pressure overload in the endocardium and the cardiac endothelium. Moreover, BMX is expressed in several types of cancers and very recently has been shown to mediate the survival and tumorigenicity of glioblastoma cancer stem cells. In the inflammatory response BMX regulates the secretion of proinflammatory cytokines induced by TNFα, IL-1β, and TLR agonists. It is required for the activation of the MAP kinase and NFκB pathways and acts at the level of the essential TAK1/TAB complex. Cellular regulation of the IL-8 promoter by BMX is dependent on membrane localization mediated by its pleckstrin homology domain, as well as on BMX kinase activity. BMX deficiency confers protection from arthritis in a mouse model known to be dependent on macrophages and IL-1β. Genetic replacement of BMX with a kinase-inactive allele surprisingly restored susceptibility to arthritis, suggesting that in vivo BMX kinase activity can be dispensable. This review summarizes recent advances in the knowledge of BMX biology and their relevance for translational medicine.

Cancer stem cell labeling using poly(l-lysine)-modified iron oxide nanoparticles

Cell labeling using magnetic nanoparticles is an increasingly used approach in noninvasive behavior tracking, in vitro separation of cancer stem cells (CSCs), and CSC-based research in cancer therapy. However, the impact of magnetic labeling on the biological properties of targeted CSCs, such as self-renewal, proliferation, multi-differentiation, cell cycle, and apoptosis, remains elusive. The present study sought to explore the potential effects on biological behavior when CSCs are labeled with superparamagnetic iron oxide (SPIO) nanoparticles in vitro. The glioblastoma CSCs derived from U251 glioblastoma multiforme were labeled with poly(l-lysine) (PLL)-modified γ-Fe2O3 nanoparticles. The iron uptake of glioblastoma CSCs was confirmed through prussian blue staining, and was further quantified using atomic absorption spectrometry. The cellular viability of the SPIO-labeled glioblastoma CSCs was assessed using a fluorescein diacetate and propidium iodide double-staining protocol. The expressed specific markers and multi-differentiation of SPIO-labeled glioblastoma CSCs were comparatively assessed by immunocytochemistry and semi-quantitative RT-PCR. The effects of magnetic labeling on cell cycle and apoptosis rate of glioblastoma CSCs and their differentiated progenies were assayed using a flow cytometer. The results demonstrated that the cell viability and proliferation capacity of glioblastoma CSCs and their differentiated progenies were not affected by SPIO labeling compared with their unlabeled counterparts. Moreover, the magnetically labeled CSCs displayed an intact multi-differentiation potential, and could be sub-cultured to form new tumor spheres, which indicates the CSCs capacity for self-renewal. In addition, cell cycle distribution, apoptosis rate of the magnetically labeled glioblastoma CSCs, and their differentiated progenies were not impaired. Therefore, the SPIO-labeled CSCs could be a feasible approach in conducting further functional analysis of targeted CSCs.

Knockdown of Ubiquitin Ligases in Glioblastoma Cancer Stem Cells Leads to Cell Death and Differentiation

The cancer stem cell (CSC) hypothesis proposes that a subpopulation of CSCs is frequently responsible for chemotherapy resistance and metastasis and is now a point of attack for research into the next generation of therapeutics. Although many of these agents are directed at inducing CSC apoptosis (as well as the bulk tumor), some agents may also decrease cell “stemness” possibly through induction of differentiation. Ubiquitin ligases, critical to virtually all cellular signaling systems, alter the degradation or trafficking of most proteins in the cell, and indeed broad perturbation of this system, through inhibition of the proteosome, is a successful cancer treatment. The authors examined several glioblastoma stem cell isolates pre- and postdifferentiation to elucidate the phenotypic effects following shRNA knockdown of ubiquitin ligases. The results were analyzed using high-content imaging (HCI) and identified ubiquitin ligases capable of inducing both CSC differentiation and apoptosis. Quite often these effects were specific to CSCs, as ubiquitin ligase knockdown in terminally differentiated progeny yielded markedly different results. The resolution of HCI at the subpopulation level makes it an excellent tool for the analysis of CSC phenotypic changes induced by shRNA knockdown and may suggest additional methods to target these cells for death or differentiation.

Glioblastoma cancer stem cells: Basis for a functional hypothesis

GBM Cancer stem cells (CSCs) are responsible for growth, recurrence and resistance to chemo- and radio-therapy. They are supposed to originate from the transformation of Neural stem cells (NSC) of the Sub-ventricular zone (SVZ) or Sub-granular zone (SGZ) of hippocampus. Alternatively, they can be the expression of a functional status of competence or dedifferentiated cells of the tumor re-acquiring stemness properties. The origin of gliomas has been put in relation with the primitive neuroepithelial cells of the SVZ or NSC or progenitors, as showed by the development of experimental tumors in rats by transplacental ethylnitrosourea administration. The demonstration of CSCs in GBM is based on Neurosphere (NS) and Adherent cell (AC) development in culture. NS share the same genetic alterations with primary tumors and express stemness antigens, whereas AC show differentiation antigens. NS are generated by the most malignant areas of GBM. CSCs are considered at the top of a hierarchy of tumor cells of which the most immature are Nestin+/CD133– cells or established on the basis of EGFR amplification or delta-EGFR. NS in serum conditions differentiate and give origin to AC, the real nature of which is still a matter of discussion. Cells in culture could be simply in vitro entities depending on culture methodology. CSCs in GBM could be tumor cells at the end of a dedifferentiation process re-acquiring stemness properties, in an opposite way to what is realized in normal cytogenesis, where stemness is lost progressively with cell differentiation. This interpretation could fit with the origin of the two GBM types, primary and secondary. In primary GBM the tumor originates directly from stem cells or progenitors from SVZ with an accelerated transformation, whereas secondary GBM originates by transformation from astrocytomas arisen through a slow transformation from migrating stem cells and progenitors.

Identification of cancer stem cells from human glioblastomas: growth and differentiation capabilities and CD133/prominin‐1 expression

CD133 can be a marker of tumorigenic CSCs (cancer stem cells) in human GBM (glioblastoma multiforme), although tumorigenic CD133-negative CSCs have been also isolated. Additional evidence indicates that CSCs from GBM exhibit different phenotypes, with increasing interest in the potential significance of the different CSCs with respect to diagnosis, prognosis and the development of novel targets for treatment. We have analysed the expression of CD133 in freshly isolated cells from 15 human GBM specimens. Only 4 of them contained cells positive for AC133 by FACS analysis, and all of them yielded distinct CSC lines, whereas only 6 CSC lines were obtained from the other 11 GBMs. Of these 10 CSCs lines, we further characterized 6 CSC lines. Three CSCs grew as fast-growing neurospheres with higher clonogenic ability, whereas the remaining 3 grew as slow-growing semi-adherent spheres of lower clonogenicity. In addition, the former CSC lines displayed better differentiation capabilities than the latter ones. PCR and Western blot analysis showed that all 6 GBM CSC lines expressed CD133/prominin-1, suggesting that cells negative by FACS analysis may actually represent cells expressing low levels of CD133 undetected by FACS. Nevertheless, all the 6 CSC lines were tumorigenic in nude mice. In conclusion, CSCs from human primary GBMs show different phenotypes and variable levels of CD133 expression, but these parameters did not directly correlate with the tumorigenic potential.

CLIC1 Functional Expression in the Plasma Membrane Correlates with Human Glioblastoma Aggressiveness

Glioblastomas are brain tumors composed by two cell types: cancer stem cells (CSCs), which are a small population able to self-renew and generate progeny, and bulk cells, consisting of a larger population committed to a precise fate. Glioblastomas are aggressive tumors because of CSC brain infiltration efficiency and their resistance to chemotherapies. Therefore, CSCs represent the most tumorigenic component of glioblastomas. Several forms of glioblastomas exhibit a level of Chloride Intracellular Channel 1 (CLIC1) expression higher than in normal brains. CLIC1 mainly localizes in the cytoplasm and in the nucleoplasm and, in stress conditions, it is able to translocate into plasma and nuclear membranes where it acts as a Cl- permeability. Four human glioblastoma biopsies were cultured in a CSCs selecting medium. By knocking down CLIC1 expression using siRNA lentiviral infection (siCLIC1), we found that CLIC1-deficient cells migrated about 50% less efficiently than cells treated with siRNA for luciferase (siLUC, control) in Boyden chamber assays. To determine whether this phenotype results from the lack of CLIC1 plasma membrane expression, CLIC1-mediated currents were estimated by using a specific inhibitor (IAA94, 100 μM) in voltage clamp experiments from perforated patches. We consistently recorded an IAA94-sensitive current in siLUC cells that was absent in siCLIC1 cells. Moreover, in non-treated CSCs we estimated the relative abundance of CLIC1-mediated current compared to the other Cl- currents in the cell. By calculating the ratio of IAA94-sensitive current over DIDS-sensitive current we found a positive correlation between tumor aggressiveness and the relative abundance of CLIC1-mediated currents in the four human glioblastomas analyzed. Our experiments highlight the involvement of CLIC1 protein in the spreading of the human glioblastoma CSCs, suggesting this protein as a possible pharmacological target to contrast the invasiveness of the brain tumor.

Dose‐dependent proteomic analysis of glioblastoma cancer stem cells upon treatment with γ‐secretase inhibitor

Notch signaling has been demonstrated to have a central role in glioblastoma (GBM) cancer stem cells (CSCs) and we have demonstrated recently that Notch pathway blockade by γ-secretase inhibitor (GSI) depletes GBM CSCs and prevents tumor propagation both in vitro and in vivo. In order to understand the proteome alterations involved in this transformation, a dose-dependent quantitative mass spectrometry (MS)-based proteomic study has been performed based on the global proteome profiling and a target verification phase where both Immunoassay and a multiple reaction monitoring (MRM) assay are employed. The selection of putative protein candidates for confirmation poses a challenge due to the large number of identifications from the discovery phase. A multilevel filtering strategy together with literature mining is adopted to transmit the most confident candidates along the pipeline. Our results indicate that treating GBM CSCs with GSI induces a phenotype transformation towards non-tumorigenic cells with decreased proliferation and increased differentiation, as well as elevated apoptosis. Suppressed glucose metabolism and attenuated NFR2-mediated oxidative stress response are also suggested from our data, possibly due to their crosstalk with Notch Signaling. Overall, this quantitative proteomic-based dose-dependent work complements our current understanding of the altered signaling events occurring upon the treatment of GSI in GBM CSCs.

Direct In Vivo Evidence for Tumor Propagation by Glioblastoma Cancer Stem Cells

High-grade gliomas (World Health Organization grade III anaplastic astrocytoma and grade IV glioblastoma multiforme), the most prevalent primary malignant brain tumors, display a cellular hierarchy with self-renewing, tumorigenic cancer stem cells (CSCs) at the apex. While the CSC hypothesis has been an attractive model to describe many aspects of tumor behavior, it remains controversial due to unresolved issues including the use of ex vivo analyses with differential growth conditions. A CSC population has been confirmed in malignant gliomas by preferential tumor formation from cells directly isolated from patient biopsy specimens. However, direct comparison of multiple tumor cell populations with analysis of the resulting phenotypes of each population within a representative tumor environment has not been clearly described. To directly test the relative tumorigenic potential of CSCs and non-stem tumor cells in the same microenvironment, we interrogated matched tumor populations purified from a primary human tumor transplanted into a xenograft mouse model and monitored competitive in vivo tumor growth studies using serial in vivo intravital microscopy. While CSCs were a small minority of the initial transplanted cancer cell population, the CSCs, not the non-stem tumor cells, drove tumor formation and yielded tumors displaying a cellular hierarchy. In the resulting tumors, a fraction of the initial transplanted CSCs maintained expression of stem cell and proliferation markers, which were significantly higher compared to the non-stem tumor cell population and demonstrated that CSCs generated cellular heterogeneity within the tumor. These head-to-head comparisons between matched CSCs and non-stem tumor cells provide the first functional evidence using live imaging that in the same microenvironment, CSCs more than non-stem tumor cells are responsible for tumor propagation, confirming the functional definition of a CSC.

Abstract B44: Therapeutic response of primary glioblastoma cancer stem cells relative to patient-matched non-stem tumor cells

Abstract Background: Populations of cells within glioblastoma multiforme (GBM) tumors that have properties similar to neural stem cells are thought to be the source of tumor cell self-renewal. These cancer stem cells (CSCs) may also be particularly resistant to therapy, although support for this important concept is limited. We asked whether early passage primary CSC cultures respond differently than patient-matched non-CSC cultures to both standard-of-care (Temozolomide (TMZ) and Radiation (XRT)) and experimental epigenetic therapy targeting histone deactylases (HDACs). Experimental Design: We established primary cultures of CSCs as adherent glioma neural stem cell and patient-matched non-CSC monolayer cultures in serum from 10 primary glioblastomas. To test response to standard glioma therapy, we treated early passage cultures with single or multiple physiological doses of TMZ (5–50uM), with or without XRT (2Gy). To examine the relative efficacy of the HDAC inhibitor vorinostat, the paired cultures were treated with physiological doses (1–2uM). Response to therapy was assayed by cell cycle analysis on a BD FACSCalibur and for cell viability using CellTiter-Glo. MGMT methylation was assessed by methylation-specific PCR and genome-wide methylation analysis was performed on IIIumina Infinium arrays. Results: The majority of patient-derived primary cultures, including CSC and non-CSC cell lines, are resistant to clinically relevant doses of TMZ. This result is in line with the general lack of response in most but not all GBM patients. Furthermore, the in vitro response to TMZ correlated with the methylation status of MGMT, a robust prognostic marker of survival in GBM patients treated with TMZ. Surprisingly, we observed for two pairs of patient-matched cultures that the CSC cultures were more responsive to single and multi-fraction XRT treatment than the non-CSCs. To further understand this difference in response in patient-matched cultures, we performed genome-wide DNA methylation analysis and found over 1500 genes with differential methylation between CSC and non-CSC cultures, including PTEN and CDKN2A. We examined the status of CD133, a marker of stemness, for our CSC and non-CSC cultures and found that cultures that were enriched for CD133+ cells were not more resistant to either TMZ or XRT. Finally, we examined whether vorinostat, an HDAC inhibitor currently in clinical trials with TMZ/XRT for GBM patients, could sensitize cultures to standard of care therapy. Clinically relevant doses of vorinostat led to decreased cell viability but did not appear to have a synergistic effect when combined with either TMZ or XRT. Conclusions: Our study demonstrates that early passage primary gliomas cultures can be used to assess drug response in less time than the interval between surgery and onset of adjuvant therapy. Furthermore, we find that for our patient-matched cultures, the CSC population is not uniformly more resistant to therapy in vitro than the non-CSC population. If the in vitro response mirrors patient response in vivo, the in vitro cultures might be beneficial for making clinical decisions on a patient-specific basis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B44.

Differential profiling studies of N‐linked glycoproteins in glioblastoma cancer stem cells upon treatment with γ‐secretase inhibitor

We have recently demonstrated that Notch pathway blockade by γ-secretase inhibitor (GSI) depletes cancer stem cells (CSCs) in Glioblastoma Multiforme (GBM) through reduced proliferation and induced apoptosis. However, the detailed mechanism by which the manipulation of Notch signal induces alterations on post-translational modifications such as glycosylation has not been investigated. Herein, we present a differential profiling work to detect the change of glycosylation pattern upon drug treatment in GBM CSCs. Rapid screening of differential cell surface glycan structures has been performed by lectin microarray on live cells followed by the detection of N-linked glycoproteins from cell lysates using multi-lectin chromatography and label-free quantitative mass spectrometry analysis. A total of 51 and 52 glycoproteins were identified in the CSC- and GSI-treated groups, respectively, filtered by a combination of decoy database searching and Trans-Proteomic Pipeline (TPP) processing. Although no significant changes were detected from the lectin microarray experiment, 7 differentially expressed glycoproteins with high confidence were captured after the multi-lectin column including key enzymes involved in glycan processing. Functional annotations of the altered glycoproteins suggest a phenotype transformation of CSCs toward a less tumorigenic form upon GSI treatment.

Autophagy Interplay with Apoptosis and Cell Cycle Regulation in the Growth Inhibiting Effect of Resveratrol in Glioma Cells

Prognosis of patients with glioblastoma (GBM) remains very poor, thus making the development of new drugs urgent. Resveratrol (Rsv) is a natural compound that has several beneficial effects such as neuroprotection and cytotoxicity for several GBM cell lines. Here we evaluated the mechanism of action of Rsv on human GBM cell lines, focusing on the role of autophagy and its crosstalk with apoptosis and cell cycle control. We further evaluated the role of autophagy and the effect of Rsv on GBM Cancer Stem Cells (gCSCs), involved in GBM resistance and recurrence. Glioma cells treated with Rsv was tested for autophagy, apoptosis, necrosis, cell cycle and phosphorylation or expression levels of key players of these processes. Rsv induced the formation of autophagosomes in three human GBM cell lines, accompanied by an upregulation of autophagy proteins Atg5, beclin-1 and LC3-II. Inhibition of Rsv-induced autophagy triggered apoptosis, with an increase in Bax and cleavage of caspase-3. While inhibition of apoptosis or autophagy alone did not revert Rsv-induced toxicity, inhibition of both processes blocked this toxicity. Rsv also induced a S-G2/M phase arrest, accompanied by an increase on levels of pCdc2(Y15), cyclin A, E and B, and pRb (S807/811) and a decrease of cyclin D1. Interestingly, this arrest was dependent on the induction of autophagy, since inhibition of Rsv-induced autophagy abolishes cell cycle arrest and returns the phosphorylation of Cdc2(Y15) and Rb(S807/811), and levels of cyclin A, and B to control levels. Finally, inhibition of autophagy or treatment with Rsv decreased the sphere formation and the percentage of CD133 and OCT4-positive cells, markers of gCSCs. In conclusion, the crosstalk among autophagy, cell cycle and apoptosis, together with the biology of gCSCs, has to be considered in tailoring pharmacological interventions aimed to reduce glioma growth using compounds with multiple targets such as Rsv.

Abstract LB-104: The histone deacetylase SIRT2 regulates the cell proliferation and tumorigenicity of glioblastoma cancer stem cells

Abstract Despite huge efforts, glioblastoma has remained one of the most intractable tumors. According to the cancer stem cell theory, the aggressive phenotype and poor prognosis of glioblastoma are attributed to the rare tumor cells called glioblastoma cancer stem cells (GSCs), which are poorly differentiated and share features with neural stem cells. However, the molecular mechanisms underlying the tumorigenicity and stemness of GSCs are still to be elucidated. In this study, we performed an RNA-interference (RNAi)-based screen and identified 29 genes that positively regulate the expression of CD133. Among these, we focused on the histone deacetylase SIRT2. When the function of SIRT2 was inhibited by RNAi or the specific inhibitor AGK2, sphere-forming capacity and cell proliferation were significantly decreased. In addition, knockdown of SIRT2 induced apoptosis in GSCs but not in “serum-differentiated” GSCs, suggesting a stem-cell specific function of SIRT2. Furthermore, GSCs in which SIRT2 expression was stably repressed by a specific small hairpin RNA (shRNA) showed impaired tumorigenicity when transplanted into immunocompromised mice. Taken together, these results imply that SIRT2 is a possible therapeutic target for glioblastoma and the existing SIRT2 inhibitors should be useful seed compounds for novel antitumor drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-104. doi:10.1158/1538-7445.AM2011-LB-104

Abstract 3397: Validation of targets involved in supporting the stem cell niche in glioblastoma multiforme

Abstract Introduction: Glioblastoma multiforme (GBM) is a cytologically malignant tumour of the central nervous system, associated with poor prognosis and fatal outcome (5 year survival, <6%). GBM cancer stem cells (CSCs), which can be identified using the immunocytochemical marker CD133, are associated with poor prognosis. The gastrin receptor, CCK2R, is also expressed in glioblastoma cell-lines and promotes cancer progression. In this study we investigated the functional role of these two molecules in GBM to elucidate any potential therapeutic benefits. Materials and methods: A panel of glioblastoma cells was grown either as monolayers, or, to provide a 3D in vitro tumour model, as neurospheres, which enrich for the cancer stem cell phenotype. Quantitative RT-PCR was used to quantify CD133 and CCK-2R gene expression over time in these cultures, and flow cytometry (FACS) to investigate expression at the protein level. CD133 and CCK-2R promoter reporters were constructed in pGL4 and promoter activity quantified using luciferase assays. Gene knockdown or specific inhibitors were used to investigate the role of these genes in glioblastoma cell tumorigenicity in vitro (using MTT, wound-healing, and neurosphere formation) and in an in vivo xenograft model. Results: CD133 and CCK-2R gene and protein expression were higher in the more tumorigenic glioblastoma cell-line, U251, compared with the less tumorigenic line, U373. For both markers, higher gene expression correlated with higher promoter activity and FACS demonstrated the presence of a small population of positively-stained cells. Expression of both genes was upregulated during neurosphere formation, which was accelerated in hypoxia. Knockdown of CD133 and CCK-2R inhibition in cell line U251 reduced neurosphere-forming potential (p<0.05). Knockdown of CD133 also significantly reduced migration (p<0.001) and increased susceptibility to the chemotherapeutic agent etoposide (p<0.05). Conclusions: CD133 and CCK-2R expression correlate with tumorigenic potential of GBM cells and are upregulated during neurosphere formation, which enriches for cancer stem cells. CD133 knockdown and CCK-2R inhibition reduced neurosphere formation. In addition, CD133 knockdown reduced GBM invasive capacity, and increased resistance to chemotherapeutic drugs. Therefore, use of CD133 and CCK-2R-targeted therapies, in combination with established standard of care may improve GBM patient outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3397. doi:10.1158/1538-7445.AM2011-3397

Abstract 3306: Screening potential drug candidates for treatment of glioblastoma patients utilizing an in-vivo mouse/ rat model system

Abstract Cancer stem cells (CSCs) are thought to be critical for engraftment and long-term growth of many tumors, including glioblastoma multiforme (GBM). The cells are at least partially spared by traditional chemotherapies and radiation therapies, and finding new treatments that can target CSCs may be critical for improving patient survival. Human glioblastoma multiforme is a heterogenous tumor composed from tumor cells and small portion of cancer stem cells, which have a high tumorigenic potential and a low proliferation rate. Glioblastoma cancer stem cells are phenotypically similar to the normal stem cells, they express CD133 gene and other genes characteristic of neural stem cells and posses self-renewal potential. The CD133+ GBM CSCs have been isolated with Celprogen Media and ECM and characterized as chemo-/radio-resistant tumor-initiating cells which are responsible as one of the many factors involved in post-treatment recurrence. In order to explore the molecular properties of tumorigenic CD133+ GBM CSCs that resist treatment, we isolated CD133+ GBM CSCs from tumors that are recurrent and have previously received chemo-/radio-therapy. We found that the purified CD133+ GBM CSCs sorted from the CD133+ GBM CSC spheres express SOX2,CD44 and are capable of clonal self-renewal. We generated in-vivo model systems in mice and rats to screen potential drug agents for the treatment of GBM patients. We were capable of generating brain tumors in mice and rats within 20 days by subcutaneously injecting 1000 GBM CSCs at the hind limbs of the rodents. Within day 5 the GBM CSCs were already present in the brain's of the rodents. At days 10, 15 & 20 the GBM CSCs labeled with GFP were located in the brain. We tested various compound candidates in this model system and we were able to show decrease in the migration of the GBM CSCs in the brain when we started the treatment at day 10 compared to non-treatment groups. We were able to demonstrate all the gene signatures associated with GBM CSCs in this model system. We do see a down regulation of certain cancer promotes genes in the treatment group when compared to non-treatment groups. The drug candidates were tested for TGFβ and STAT pathways. The chemotherapeutic agents tested were Among the 300 compounds tested Adria, Taxol, DTIC, Thalidomide, Paclitaxel, Abraxane, Ertibux and Velcade. We were able to show 4 compounds that were effective against GBM CSC targeting selected pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3306. doi:10.1158/1538-7445.AM2011-3306

Abstract 126: Characterization of microRNA-10b in human glioblastoma

Abstract Glioblastoma is the most common and deadly malignant brain tumor. One main reason of resistance of glioblastoma to therapy is the ability of the tumor cells to migrate and invade the surrounding brain, rendering total surgical removal of the tumor practically impossible. MicroRNAs are recently discovered small noncoding regulatory RNA molecules with emerging critical roles in cancer. MicroRNAs modulate protein expression by binding to the 3′-untranslated region of mRNA and promoting RNA degradation or inhibiting transcription. By acting on oncogenes and tumor suppressors, microRNAs can behave as tumor suppressors or oncogenes, respectively. In this study, we show that microRNA-10b (miR-10b) is deregulated in glioblastoma where it regulates tumor cell migration and invasion. While analyzing The Cancer Genome Atlas (TCGA) data, we noticed that miR-10b is associated with poor prognosis of glioblastoma patients. We therefore embarked on a comprehensive characterization of miR-10b in glioblastoma. We first assessed the expression of miR-10b in human glioblastoma cells and cancer stem cells using quantitative RT-PCR. We found that miR-10b expression is up-regulated in all human glioblastoma cell lines and glioblastoma stem cells tested as compared to normal human astrocytes. We then tested the effects of miR-10b inhibition on cell cycle status, cell proliferation, cell invasion, cell migration, clonogenicity and apoptosis by propidium iodide flow cytometry, cell counting, transwell invasion assay, scratch assay, soft agar assay and annexin V flow cytometry, respectively. Glioblastoma cells, stem cells or normal human astrocytes were transfected with a specific anti-miR-10b antagomir and assessed for the above functional endpoints. Inhibition of miR-10b had variable effects on cell proliferation. It only mildly decreased the proliferation of established glioblastoma cell lines but exhibited greater inhibition on the proliferation of glioblastoma stem cells. Inhibition of miR-10b did not affect apoptosis or death of any of the tested cells. Importantly, inhibition of miR-10b strongly inhibited cell invasion, cell migration and anchorage-independent growth in glioblastoma cells and stem cells. Conversely, overexpression of miR-10b by transfection of pre-miR-10b in glioblastoma cells promoted tumor cell invasion and migration in vitro. We are currently in the process of researching potential targets of miR-10b that mediate its effects on glioblastoma cell migration and invasion. We are also studying the potential correlation between the levels of miR-10b in human glioblastoma tissues and tumor grade. Altogether, our findings suggest that miR-10b is a tumor promoter that is upregulated in glioblastoma where it induces tumor cell migration and invasion. These preliminary results suggest that targeting miR-10b could be a promising new strategy for glioblastoma therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 126. doi:10.1158/1538-7445.AM2011-126

1H-Nuclear Magnetic Resonance Spectroscopy of Glioblastoma Cancer Stem Cells

The noninvasive tracking of glioblastoma cancer stem cells (CSCs) in vivo constitutes a prerequisite for the development of CSC-specific therapies. Therefore, as a pilot study to identify CSC biomarkers for clinical magnetic resonance spectroscopy, 10 CSC lines were investigated using high-resolution (1)H-nuclear magnetic resonance ((1)H-NMR) spectroscopy at 600 and 800 MHz (14.4 and 18.8 T) under reproducible in vitro conditions. The spectra were analyzed using principal component analysis (PCA), and spectral regions of high variability were evaluated regarding correlations to stem cell-related properties (clonogenic index and CD133 positivity) and cell death. PCA revealed that duplicates of CSC lines clustered together suggesting a characteristic (1)H-NMR pattern of each CSC line. PCA enabled discrimination between samples with high and low clonogenicity, that is, clustering according to one of the hallmarks of stemness in samples with high viability. High/moderate correlations to clonogenicity and CD133 were found in spectral regions with high variability. In contrast, the mobile lipid signal at 1.28 ppm correlated to cell death, but not to stemness, as published previously for neural progenitor cells. In conclusion, our exploratory study demonstrates the correlation of specific resonances within (1)H-NMR spectra with stem cell properties and advocates the use of the 1.28 ppm resonance as biomarker for cell death also in CSCs.