GBM-derived Cells Research Articles

Proteomic Analysis Implicates Vimentin in Glioblastoma Cell Migration.

We previously showed lithium chloride (LiCl) and other inhibitors of glycogen synthase kinase-3 (GSK-3) including 6-bromo-indirubin-3-oxime (BIO), can block glioblastoma (GBM) cell migration. To investigate the mechanisms involved we used two-dimensional difference in-gel electrophoresis (2D-DIGE) and mass spectrometry to identify proteins altered after treatment of U251 GBM cells with 20 mM LiCl. Downregulation of the intermediate filament protein vimentin was the most significant change identified. Analysis of patient tumor samples revealed that vimentin is expressed abundantly in GBM, and is prognostic especially in lower grade tumors. Additionally, siRNA-mediated vimentin knockdown impaired GBM migration. Western blotting showed that treatment with LiCl or small molecule GSK-3 inhibitors led to the rapid downregulation of detergent soluble vimentin levels across a panel of GBM-derived cells. Fluorescence reactivation after photobleaching (FRAP) microscopy studies showed a significant reduction in the ability of the vimentin cytoskeleton to recover from photo-bleaching in the presence of LiCl or BIO. Biochemical studies revealed that GSK-3 and vimentin directly interact, and analysis of vimentin revealed a GSK-3 consensus phosphorylation site. We conclude that anti-migratory compounds with the ability to inhibit GSK-3 have effects on vimentin cytoskeletal dynamics, which may play a role in their anti-invasive activity.

Wnt and Notch signaling govern self-renewal and differentiation in a subset of human glioblastoma stem cells.

Developmental signal transduction pathways act diversely, with context-dependent roles across systems and disease types. Glioblastomas (GBMs), which are the poorest prognosis primary brain cancers, strongly resemble developmental systems, but these growth processes have not been exploited therapeutically, likely in part due to the extreme cellular and genetic heterogeneity observed in these tumors. The role of Wnt/βcatenin signaling in GBM stem cell (GSC) renewal and fate decisions remains controversial. Here, we report context-specific actions of Wnt/βcatenin signaling in directing cellular fate specification and renewal. A subset of primary GBM-derived stem cells requires Wnt proteins for self-renewal, and this subset specifically relies on Wnt/βcatenin signaling for enhanced tumor burden in xenograft models. In an orthotopic Wnt reporter model, Wnthi GBM cells (which exhibit high levels of βcatenin signaling) are a faster-cycling, highly self-renewing stem cell pool. In contrast, Wntlo cells (with low levels of signaling) are slower cycling and have decreased self-renewing potential. Dual inhibition of Wnt/βcatenin and Notch signaling in GSCs that express high levels of the proneural transcription factor ASCL1 leads to robust neuronal differentiation and inhibits clonogenic potential. Our work identifies new contexts for Wnt modulation for targeting stem cell differentiation and self-renewal in GBM heterogeneity, which deserve further exploration therapeutically.

Determinants for Effective ALECSAT Immunotherapy Treatment on Autologous Patient-Derived Glioblastoma Stem Cells

Glioblastoma (GBM) is the most aggressive primary brain tumor with a median survival of less than 15 months, emphasizing the need for better treatments. Immunotherapy as a treatment for improving or aiding the patient’s own immune defense to target the tumor has been suggested for GBM. A randomized clinical trial of adoptive cell transfer using ALECSAT (Autologous Lymphoid Effector Cells Specific Against Tumor Cells) is currently ongoing in Sweden. Here we performed a paired pre-clinical study to investigate the composition and in vitro effect of ALECSAT and identify determinants for the effect using autologous GBM-derived cancer stem cells (CSC), immunocytochemistry and flow cytometry. We show a clear dose-response relationship of ALECSAT on CSC, suggesting that the number of infused cells is of importance. In addition, the in vitro effect of ALECSAT on CSC correlated significantly to the blood count of T helper (Th) cells in the patient indicating a potential benefit of collecting cells for ALECSAT preparation at an even earlier stage when patients generally have a better blood count. The factors identified in this study will be important to consider in the design of future immunotherapy trials to achieve prolonged survival.

Pediatric glioblastoma cells inhibit neurogenesis and promote astrogenesis, phenotypic transformation and migration of human neural progenitor cells within cocultures

Neural progenitor cell (NPC) fate is influenced by a variety of biological cues elicited from the surrounding microenvironment and recent studies suggest their possible role in pediatric glioblastoma multiforme (GBM) development. Since a few GBM cells also display NPC characteristics, it is not clear whether NPCs transform to tumor cell phenotype leading to the onset of GBM formation, or NPCs migrate to developing tumor sites in response to paracrine signaling from GBM cells. Elucidating the paracrine interactions between GBM cells and NPCs in vivo is challenging due to the inherent complexity of the CNS. Here, we investigated the interactions between human NPCs (ReNcell) and human pediatric GBM-derived cells (SJ-GBM2) using a Transwell® coculture setup to assess the effects of GBM cells on ReNcells (cytokine and chemokine release, viability, phenotype, differentiation, migration). Standalone ReNcell or GBM cultures served as controls. Qualitative and quantitative results from ELISA®, Live/Dead® and BrdU assays, immunofluorescence labeling, western blot analysis, and scratch test suggests that although ReNcell viability remained unaffected in the presence of pediatric GBM cells, their morphology, phenotype, differentiation patterns, neurite outgrowth, migration patterns (average speed, distance, number of cells) and GSK-3β expression were significantly influenced. The cumulative distance migrated by the cells in each condition was fit to Furth's formula, derived formally from Ornstein-Uhlenbeck process. ReNcell differentiation into neural lineage was compromised and astrogenesis promoted within cocultures. Such coculture platform could be extended to identify the specific molecules contributing to the observed phenomena, to investigate whether NPCs could be transplanted to replace lesions of excised tumor sites, and to elucidate the underlying molecular pathways involved in GBM-NPC interactions within the tumor microenvironment.

PATZ1 is a new prognostic marker of glioblastoma associated with the stem-like phenotype and enriched in the proneural subtype.

Glioblastoma (GBM), the most malignant of the brain tumors, has been classified on the basis of molecular signature into four subtypes: classical, mesenchymal, proneural and neural, among which the mesenchymal and proneural subtypes have the shortest and longest survival, respectively. Here we show that the transcription factor PATZ1 gene is upregulated in gliomas compared to normal brain and, among GBMs, is particularly enriched in the proneural subtype and co-localize with stemness markers. Accordingly, in GBM-derived glioma-initiating stem cells (GSCs) PATZ1 is overexpressed compared to differentiated tumor cells and its expression significantly correlates with the characteristic stem cell capacity to grow as neurospheres in vitro. Interestingly, survival analysis demonstrated that PATZ1 lower levels informed poor prognosis in GBM and, specifically, in the proneural subgroup, suggesting it may serve a role as diagnostic and prognostic biomarker for intra-subtype heterogeneity of proneural GBM. We also show that PATZ1 suppresses the expression of the mesenchyme-inducer CXCR4, and that PATZ1 and CXCR4 are inversely correlated in GSC and proneural GBM. Overall these findings support a central role of PATZ1 in regulating malignancy of GBM.

Dual treatment with shikonin and temozolomide reduces glioblastoma tumor growth, migration and glial-to-mesenchymal transition.

Glioblastomas (GBM) comprise 17% of all primary brain tumors. These tumors are extremely aggressive due to their infiltrative capacity and chemoresistance, with glial-to-mesenchymal transition (GMT) proteins playing a prominent role in tumor invasion. One compound that has recently been used to reduce the expression of these proteins is shikonin (SHK), a naphthoquinone with anti-tumor properties. Temozolomide (TMZ), the most commonly used chemotherapeutic agent in GBM treatment, has so far not been studied in combination with SHK. Here, we investigated the combined effects of these two drugs on the proliferation and motility of GBM-derived cells. The cytotoxic and proliferative effects of SHK and TMZ on human GBM-derived cells were tested using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Ki67 staining and BrdU incorporation assays. The migration capacities of these cells were evaluated using a scratch wound assay. The expression levels of β3 integrin, metalloproteinases (MMPs) and GMT-associated proteins were determined by Western blotting and immunocytochemistry. We found that GBM-derived cells treated with a combination of SHK and TMZ showed decreases in their proliferation and migration capacities. These decreases were followed by the suppression of GMT through a reduction of β3 integrin, MMP-2, MMP-9, Slug and vimentin expression via inactivation of PI3K/AKT signaling. From our results we conclude that dual treatment with SHK and TMZ may constitute a powerful new tool for GBM treatment by reducing therapy resistance and tumor recurrence.

ID2 promotes survival of glioblastoma cells during metabolic stress by regulating mitochondrial function

Tumor cells proliferate in cellular environments characterized by a lack of optimal tissue organization resulting oftentimes in compromised cellular metabolism affecting nutrition, respiration, and energetics. The response of tumor cells to adverse environmental conditions is a key feature affecting their pathogenicity. We found that inhibitor of DNA binding 2 (ID2) expression levels significantly correlate with the ability of glioblastoma (GBM)-derived cell lines to survive glucose deprivation. ID2 suppressed mitochondrial oxidative respiration and mitochondrial ATP production by regulating the function of mitochondrial electron transport chain (mETC) complexes, resulting in reduced superoxide and reactive oxygen species (ROS) production from mitochondria. ID2 suppression of ROS production reduced mitochondrial damage and enhanced tumor cell survival during glucose deprivation. Bioinformatics analysis of GBM gene expression data from The Cancer Genome Atlas (TCGA) database revealed that expression of ID2 mRNA is unique among ID gene family members in correlating with the expression of nuclear genes involved in mitochondrial energy metabolism and assembly of mETC. Our data indicate that the expression level of ID2 in GBM cells can predict the sensitivity of GBM-derived tumor cells to decreased glucose levels. Low levels of ID2 expression in human GBM tissues may identify a clinical group in which metabolic targeting of glycolytic pathways can be expected to have the greatest therapeutic efficacy.

Platelets from glioblastoma patients promote angiogenesis of tumor endothelial cells and exhibit increased VEGF content and release

Glioblastoma multiforme (GBM) is the most common and fatal intracranial cancer in humans and exhibits intense and aberrant angiogenesis that sustains its malignancy and involves several angiogenic signals. Among them, vascular endothelial growth factor (VEGF) plays a key role and is overexpressed in GBM. Different cells appear to act as triggers of the aberrant angiogenesis, and, among them, platelets act as key participants. In order to provide further insights into the platelet features and angiogenic role in GBM, this study investigated the effects of platelet releasate on GBM-derived endothelial cells (GECs) and the levels of VEGF and endostatin, as pro- and anti-angiogenic components of platelet releasate from GBM patients. We demonstrate for the first time that: 1) platelet releasate exerts powerful pro-angiogenic effect on GECs, suggesting it might exert a role in the aberrant angiogenesis of GBM; 2) ADP and thrombin stimulation leads to significantly higher level of VEGF, but not of endostatin, in the releasate of platelets from GBM patients than those from healthy subjects; and 3) the intraplatelet concentrations of VEGF were significantly elevated in GBM patients as compared to controls. Moreover, we found a direct correlation between platelet-released VEGF and overall survival in our patient cohort. Although preliminary, these findings prompt further investigations to clarify the biologic relevance of platelet VEGF in GBM and prospective studies for screening GBM patients for anti-VEGF therapy and/or to optimize this treatment.

Accelerating glioblastoma drug discovery: Convergence of patient-derived models, genome editing and phenotypic screening.

Patients diagnosed with glioblastoma (GBM) continue to face a bleak prognosis. It is critical that new effective therapeutic strategies are developed. GBM stem cells have molecular hallmarks of neural stem and progenitor cells and it is possible to propagate both non-transformed normal neural stem cells and GBM stem cells, in defined, feeder-free, adherent culture. These primary stem cell lines provide an experimental model that is ideally suited to cell-based drug discovery or genetic screens in order to identify tumour-specific vulnerabilities. For many solid tumours, including GBM, the genetic disruptions that drive tumour initiation and growth have now been catalogued. CRISPR/Cas-based genome editing technologies have recently emerged, transforming our ability to functionally annotate the human genome. Genome editing opens prospects for engineering precise genetic changes in normal and GBM-derived neural stem cells, which will provide more defined and reliable genetic models, with critical matched pairs of isogenic cell lines. Generation of more complex alleles such as knock in tags or fluorescent reporters is also now possible. These new cellular models can be deployed in cell-based phenotypic drug discovery (PDD). Here we discuss the convergence of these advanced technologies (iPS cells, neural stem cell culture, genome editing and high content phenotypic screening) and how they herald a new era in human cellular genetics that should have a major impact in accelerating glioblastoma drug discovery.

Abstract PR04: The role of long noncoding RNA HIF1A-AS2 in hypoxic environment of glioblastoma

Abstract Purpose: While multiple protein-coding genes are known to play a crucial role in the formation and progression of glioblastoma multiforme (GBM), the role of long-non-coding RNAs (lncRNAs) in these cascades remains to be fully characterized. Considering the fundamental roles of hypoxia in the cellular and microenvironmental complexity and unexplored function of lncRNAs in GBM pathobiology, the identification of novel lncRNAs and their target pathways that drive the adaptation to hypoxic niche is crucial for better understanding of the development and progression of this highly heterogeneous brain tumor. Transcriptome profiling of GBM have revealed the presence of four clearly distinguishable subtypes, variably expressed in individual cells within a tumor. This finding may have potential clinical implications, including subtype-specific rearrangements of transcriptional programs related to oncogenic signaling, growth and hypoxia. However, the subtype-specific role of lncRNAs in tumorigenic potential of GBM subtypes remains largely unknown. Materials and Methods: The brain tissue samples including GBM tumors and non-pathological tissue adjacent to the tumor were collected. GBM-derived primary stem cells (GSCs) classified by transcriptome analysis as proneural (P) and mesenchymal (M), were collected and exposed to hypoxic conditions. Using custom designed Nanostring platform analysis followed by qPCR, the expression patterns of 70 cancer-related lncRNAs were characterized. The in situ hybridization and qPCR analysis was used to validate sub-cellular localization of selected lncRNA. RNA immunoprecipitation (RIP) and mass-spectroscopy (MS) was performed to map RNA-protein interaction and identified targets were validated by Western blotting. The global GSC transcriptome profiling upon lncRNA de-regulation was performed using Pan Cancer Nanostring platform. The knock-down and overexpression strategies in GSC were used to characterize cellular and molecular phenotypes in vitro and in in vivo intracranial GBM model. Results: We identified lncRNA HIF1A-AS2 (Hypoxia Inducible Factor 1 alpha - antisense 2) as one of the most deregulated in GBM comparing to the matched adjacent tissue. Despite lack of difference in the hypoxia-dependent activation of HIF1A protein between P and M GSC, HIF1A-AS2 was specifically upregulated in M GSC in vitro and in vivo. The deregulation of expression of HIF1A-AS2 had a broad effect on autophagy-related signaling network regulated in response to hypoxia in M but not P GSC. The IGF2BP2 and DHX9 were identified as direct protein partners of this lncRNA. The deregulation of HIF1A-AS2 affected the recruitment of IGF2BP2 to its mRNA targets (e.g. HMG1) and stability of DHX9 protein (but not mRNA), resulting in deregulation of its target genes (e.g FOSL1). Downregulation of HIF1A-AS2 in vivo led to de-regulation of its downstream effectors and resulted in survival benefit of mice bearing M GSC-originated tumors. Conclusion: P and M GSC respond differently to hypoxic stress by induction of autophagy to maintain their homeostasis and viability and this mechanism is controlled by HIF1A-AS2. This abstract is also presented as Poster A16. Citation Format: Marco Mineo, Franz Ricklefs, Shawn S. Lyons, Pavel Ivanov, E. Antonio Chiocca, Jakub Godlewski, Agnieszka Bronisz. The role of long noncoding RNA HIF1A-AS2 in hypoxic environment of glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr PR04.

An intrinsic DFF40/CAD endonuclease deficiency impairs oligonucleosomal DNA hydrolysis during caspase-dependent cell death: a common trait in human glioblastoma cells.

Glioblastoma (GBM) or grade IV astrocytoma is one of the most devastating human cancers. The loss of DFF40/CAD, the key endonuclease that triggers oligonucleosomal DNA fragmentation during apoptosis, has been linked to genomic instability and cell survival after radiation. Despite the near inevitability of GBM tumor recurrence after treatment, the relationship between DFF40/CAD and GBM remains unexplored. We studied the apoptotic behavior of human GBM-derived cells after apoptotic insult. We analyzed caspase activation and the protein levels and subcellular localization of DFF40/CAD apoptotic endonuclease. DFF40/CAD was also evaluated in histological sections from astrocytic tumors and nontumoral human brain. We showed that GBM cells undergo incomplete apoptosis without generating oligonucleosomal DNA degradation despite the correct activation of executioner caspases. The major defect of GBM cells relied on the improper accumulation of DFF40/CAD at the nucleoplasmic subcellular compartment. Supporting this finding, DFF40/CAD overexpression allowed GBM cells to display oligonucleosomal DNA degradation after apoptotic challenge. Moreover, the analysis of histological slices from astrocytic tumors showed that DFF40/CAD immunoreactivity in tumoral GFAP-positive cells was markedly reduced when compared with nontumoral samples. Our data highlight the low expression levels of DFF40/CAD and the absence of DNA laddering as common molecular traits in GBM. These findings could be of major importance for understanding the malignant behavior of remaining tumor cells after radiochemotherapy.

METB-04COULD ALTERED FATTY ACID AND EICOSANOID METABOLISM PROVIDE NOVEL THERAPEUTIC TARGETS IN BRAIN TUMOURS?

Glioblastoma (GBM) is the most common and most malignant glioma of the central nervous system. Meningiomas derived from the arachnoid cap cells are less aggressive tumours but atypical and malignant meningiomas have similar migratory, invasive and highly proliferative properties to that of GBMs and are also particularly difficult to treat. Recent investigations in our laboratory have focussed on the eicosanoid pathways present in gliomas and meningiomas. Techniques used include qRT-PCR, Immunohistochemistry, Western blotting, ESI-LC-MS/MS, GCMS and cell migration assays. We have identified several of the eicosanoids involved in the control of both cell proliferation and apoptosis in glioma cells. A strong relationship was found between prostaglandins of the E series and their receptors, and cell migration in GBM-derived cells. qRT-PCR analyses have identified mRNA's correlated with differences in survival in GBM patients. GCMS and ESI-LC-MS/MS analyses, to profile the fatty acids and eicosanoids present in primary brain tumours, have shown significant differences between tumour types and among tumour grades. High grade tumours have significantly altered HODE, HETE and HEPE contents in comparison with lower grade tumours. We are currently investigating the role of eicosanoids in the control of ABC transporters in glioma cells and primary brain tumours. The expression and activity of ABCB and ABCC transporters were altered by the presence of cyclooxygenase (COX) 1 and 2 inhibitors. The improved response to chemotherapeutic drugs found after interfering with eicosanoid production supports the hypothesis that altered eicosanoid metabolism could be an important approach to adjuvant therapy, which may lead to improvements in patient response to drug treatment in the future. Financial support: FAPESP, CAPES, CNPq.

Design and synthesis of 2-oxindole based multi-targeted inhibitors of PDK1/Akt signaling pathway for the treatment of glioblastoma multiforme

Aggressive behavior and diffuse infiltrative growth are the main features of Glioblastoma multiforme (GBM), together with the high degree of resistance and recurrence. Evidence indicate that GBM-derived stem cells (GSCs), endowed with unlimited proliferative potential, play a critical role in tumor development and maintenance. Among the many signaling pathways involved in maintaining GSC stemness, tumorigenic potential, and anti-apoptotic properties, the PDK1/Akt pathway is a challenging target to develop new potential agents able to affect GBM resistance to chemotherapy. In an effort to find new PDK1/Akt inhibitors, we rationally designed and synthesized a small family of 2-oxindole derivatives. Among them, compound 3 inhibited PDK1 kinase and downstream effectors such as CHK1, GS3Kα and GS3Kβ, which contribute to GCS survival. Compound 3 appeared to be a good tool for studying the role of the PDK1/Akt pathway in GCS self-renewal and tumorigenicity, and might represent the starting point for the development of more potent and focused multi-target therapies for GBM.

Temozolomide sensitizes stem-like cells of glioma spheres to TRAIL-induced apoptosis via upregulation of casitas B-lineage lymphoma (c-Cbl) protein.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has potent antitumor effects in glioma cell lines but has shown little clinical benefit for patients. We investigated whether the widely used chemotherapeutic agent temozolomide (TMZ) can sensitize glioma stem-like cells (GSCs) from human glioblastoma multiforme (GBM) to TRAIL-induced apoptosis. GSCs were isolated from GBM, and stem cell properties were confirmed by immunocytochemistry and in vivo tumorigenicity. Primary GSCs (PGCs) were produced by serum treatment of GBM-derived cells. Changes in expression levels of various TRAIL-related signaling factors before and after TRAIL or TRAIL + TMZ treatment were measured by Western blotting. Overexpression vectors and siRNAs were used to investigate mechanism of TRAIL sensitivity. GSCs showed greater resistance to TRAIL-induced apoptosis than PGCs and had lower basal caspase activity. Caspase knockdown in PGCs reduced TRAIL sensitivity. Expression levels of c-Fas-associated death domain-like interleukin 1-converting enzyme-like inhibitory protein long and short isoforms (c-FLIPL and c-FLIPS) were significantly higher in GSCs than PGCs, and siRNA-mediated c-FLIP knockdown in GSCs enhanced TRAIL-induced apoptosis. TMZ enhanced TRAIL-induced apoptosis in GSCs and downregulated c-FLIP expression. Add of TMZ also upregulated the expression of the E3 ubiquitin ligase casitas B-lineage lymphoma (c-Cbl). Moreover, overexpression of c-Cbl alone reduced c-FLIP expression, and c-Cbl knockdown both enhanced c-FLIP expression and reduced the potentiating effect of TMZ on TRAIL-induced apoptosis. The result indicated that TMZ may overcome TRAIL resistance in GSCs by suppressing c-FLIP expression through c-Cbl-mediated ubiquitination and degradation.

Identification of Global DNA Methylation Signatures in Glioblastoma-Derived Cancer Stem Cells

Glioblastoma (GBM) is the most common and most aggressive primary brain tumor in adults. The existence of a small population of stem-like tumor cells that efficiently propagate tumors and resist cytotoxic therapy is one proposed mechanism leading to the resilient behavior of tumor cells and poor prognosis. In this study, we performed an in-depth analysis of the DNA methylation landscape in GBM-derived cancer stem cells (GSCs). Parallel comparisons of primary tumors and GSC lines derived from these tumors with normal controls (a neural stem cell (NSC) line and normal brain tissue) identified groups of hyper- and hypomethylated genes that display a trend of either increasing or decreasing methylation levels in the order of controls, primary GBMs, and their counterpart GSC lines, respectively. Interestingly, concurrent promoter hypermethylation and gene body hypomethylation were observed in a subset of genes including MGMT, AJAP1 and PTPRN2. These unique DNA methylation signatures were also found in primary GBM-derived xenograft tumors indicating that they are not tissue culture-related epigenetic changes. Integration of GSC-specific epigenetic signatures with gene expression analysis further identified candidate tumor suppressor genes that are frequently down-regulated in GBMs such as SPINT2, NEFM and PENK. Forced re-expression of SPINT2 reduced glioma cell proliferative capacity, anchorage independent growth, cell motility, and tumor sphere formation in vitro. The results from this study demonstrate that GSCs possess unique epigenetic signatures that may play important roles in the pathogenesis of GBM.

USP1 targeting impedes GBM growth by inhibiting stem cell maintenance and radioresistance.

Clinical benefits from standard therapies against glioblastoma (GBM) are limited in part due to intrinsic radio- and chemoresistance of GBM and inefficient targeting of GBM stem-like cells (GSCs). Novel therapeutic approaches that overcome treatment resistance and diminish stem-like properties of GBM are needed. We determined the expression levels of ubiquitination-specific proteases (USPs) by transcriptome analysis and found that USP1 is highly expressed in GBM. Using the patient GBM-derived primary tumor cells, we inhibited USP1 by shRNA-mediated knockdown or its specific inhibitor pimozide and evaluated the effects on stem cell marker expression, proliferation, and clonogenic growth of tumor cells. USP1 was highly expressed in gliomas relative to normal brain tissues and more preferentially in GSC enrichment marker (CD133 or CD15) positive cells. USP1 positively regulated the protein stability of the ID1 and CHEK1, critical regulators of DNA damage response and stem cell maintenance. Targeting USP1 by RNA interference or treatment with a chemical USP1 inhibitor attenuated clonogenic growth and survival of GSCs and enhanced radiosensitivity of GBM cells. Finally, USP1 inhibition alone or in combination with radiation significantly prolonged the survival of tumor-bearing mice. USP1-mediated protein stabilization promotes GSC maintenance and treatment resistance, thereby providing a rationale for USP1 inhibition as a potential therapeutic approach against GBM.

Abstract 1379: Identification of global DNA methylation signatures in glioblastoma-derived cancer stem cells

Abstract Glioblastoma (GBM) is the most common and most aggressive brain tumor in adults. Its frequent recurrence after resection and dismal prognosis are thought to be due to a small population of stem-like tumor cells that efficiently propagate tumors and resist cytotoxic therapy. In this study, we performed an in depth analysis of the DNA methylation landscape in GBM-derived cancer stem cells (GSCs). Parallel comparisons of primary GBM tumors and GSC lines derived from these tumors with normal controls (a neural stem cell (NSC) line and normal brain tissue) identified two groups hyper- and hypomethylated of genes that display a trend of either increasing or decreasing methylation levels in the order of controls, primary GBMs, and their counterpart GSC lines, respectively. Interestingly, concurrent promoter hypermethylation and gene body hypomethylation were observed in a subset of genes including MGMT, AJAP1 and PTPRN2. These unique DNA methylation signatures were also be found in primary GBM-derived xenograft tumors suggesting that they were not tissue culture-related epigenetic changes. Integration of GSC-specific epigenetic signatures with gene expression analysis further identified candidate tumor suppressor genes that are frequently down regulated in GBMs such as SPINT2, NEFM, and PENK. The comparison between the NSC line and the normal brain tissue sample leads to the identification of a group of NSC-specific differentially methylated regions (nsDMRs). Cluster analysis using nsDMRs revealed the presence of stem cell-specific DNA methylation signatures in both primary GBM and GSC lines. Higher expression of genes associated with stem cell-specific DNA methylation signatures such as DNMT3A, STAT5A, CSTB, PMEPA1 and G6PD in GBM patients was found to be associated with poor patient survival. The results from this study demonstrate the utility of using cancer stem cell models for advancing understanding of the pathobiology of gliomas. Citation Format: Eun Joon Lee, Prakash Rath, Jimei Liu, Dungsheng Ryu, Alan Free, Lirong Pei, Douglas C Anthony, Suash Sharma, Mark D Kirk, John J. Laterra, Duck Hwan Ryu, Jeong-Hyeon Choi, Huidong Shi, Douglas C. Miller, N. Scott Litofsky, Qi Feng. Identification of global DNA methylation signatures in glioblastoma-derived cancer stem cells. [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 1379. doi:10.1158/1538-7445.AM2014-1379

Abstract 3039: The MELK/FOXM1 axis is a master regulator of proneural to mesenchymal transition (PMT) in glioma stem cells by controlling EZH2 transcriptional activity

Abstract In the Central Nervous System, stem cells are present in the normal brain and primary brain tumor, glioblasotoma multiforme (GBM). Both normal and oncogenic stem cells play central roles in organogenesis and tumorigenesis, respectively. Previously we identified that the mitotic kinase MELK binds and phosphorylates the oncogenic transcription factor FOXM1 to regulate the growth of both brain-derived and GBM-derived stem cells (GSCs). Here we demonstrate that the Enhancer of Zeste Homolog 2 (EZH2), the lysine methyltransferase of Polycomb repressive complex 2, is the major transcriptional target of the MELK-FOXM1 complex in GSCs. PCR-based analysis of FOXM1 immunoprecipitated chromatin determined the binding of FOXM1 to the EZH2 promoter region to drive its transcriptional activity. This signaling is mediated through the protein complex formation of FOXM1 and MELK in a MELK kinase-dependent manner. The MELK-FOXM1 driven EZH2 signals are required to retain the immature stem cell trait and mesenchymal identity in GBM, as well as GSCs' radiation resistant phenotype in a MELK kinase-dependent manner. Clinically, GBM tumors co-express MELK and EZH2 with significant elevation in post-irradiation recurrent tumors. This post-irradiation elevation of MELK and EZH2 was observed specifically in GSCs. Irradiation induces transformation of proneural (PN) GSCs into a Mesenchymal (Mes)-like GSC phenotype (PMT) that is highly resistant to radiation treatment, and inhibition of EZH2 by shRNA reverses the radiation-resistant phenotype of Mes GSCs. Elimination of MELK or FOXM1 by shRNA reduced EZH2 expression resulting in attenuated stemness and radiation resistance in vitro and in vivo. These phenotypes were completely restored by exogenous expression of EZH2. To our knowledge, this study provides the first evidence of the role of MELK-FOXM1-EZH2 signaling axis for maintenance of stem cell trait and PMT regulation in GSCs. Identification of the role of transcriptional regulator EZH2 in PMT by the mitotic kinase MELK and transcription factor FOXM1 complex sheds lights on a novel therapeutic target for aggressive cancers including GBM. Citation Format: Kaushal Joshi, Sunghak Kim, Jeongwu Lee, Ronald Waclaw, Lisa Salcini, Ichiro Nakano. The MELK/FOXM1 axis is a master regulator of proneural to mesenchymal transition (PMT) in glioma stem cells by controlling EZH2 transcriptional activity. [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 3039. doi:10.1158/1538-7445.AM2014-3039

Growth arrest and forced differentiation of human primary glioblastoma multiforme by a novel small molecule.

Glioblastoma multiforme is the most common malignant brain tumor in adults, with an average survival of less than one year due to its resistance to therapy. Recent studies reported that GBM initiates from CD133-expressing cancer stem cells (CSC). However, the efficacy of CSC targeting is limited. A newly developed approach in cancer treatment is the forced differentiation of cancer cells. Here, we show that the treatment of the novel small molecule, CG500354, into CD133-expressing human primary GBM cells induces growth arrest by cell cycle regulators, p53, p21, p27 and phase-specific cyclins, and neural differentiation, as confirmed by neural progenitor/precursor markers, nestin, GFAP and Tuj1. When GBM-derived cells caused the tumors in NOD/SCID mice, CG500354 induced GBM-derived cells differentiation into Tuj1 and GFAP expressing cells. We next demonstrated that CG500354 plays a tumor-suppressive role via cAMP/CREB signaling pathway. CG500354 increases not only the extracellular cAMP level but also the protein level of PKA and CREB. Additionally, both mimetic substances, Forskolin and Rolipram, revealed comparable results with CG500354. Our findings indicate that induction of growth arrest and neural differentiation via cAMP/CREB signaling pathway by CG500354 treatment suggests the novel targeting of PDE4D in the development of new drugs for brain tumor therapy.

Cells isolated from human glioblastoma multiforme express progesterone-induced blocking factor (PIBF).

Glioblastoma multiforme (GBM) is the most common and malignant tumor in the central nervous system. One of the contemporary hypotheses postulates that its pathogenesis is associated with the cancer stem cells (CSCs) which originate from mutations in the normal neural stem cells residing in their specific "niches." Simultaneously with its aggressive development the tumor suppresses the local immune system by different secreted and/or cell expressed factors. Progesterone-induced blocking factor (PIBF) is an immunomodulatory protein with known role in the regulation of the immune response in the reproductive system. Expression of PIBF has been described in some tumors as one of the factors suppressing the anti-tumor immunity. The aim of the present study was to check for the expression of PIBF from cells isolated from six GBMs. To characterize the cultured cells and to study the PIBF expression confocal microscopy, flow cytometry, ELISA, and real-time PCR were used. The results obtained showed expression of markers typical for cancer CSCs and secretion of interleukin 6 by the GBM-derived cultured cells. The results convincingly prove that PIBF is intracellularly expressed by the cultured cells from the all six GBM samples, and this fact is confirmed by three different methods-flow cytometry, confocal microscopy, and real-time PCR. This paper reports for the first time the expression of PIBF by GBM-derived cells cultured in vitro and reveals a new aspect of the immunosuppressive mechanism used by GBM in escaping the immune control.