Glioma Stem-like Cells Research Articles

Regulation of OSU-03012 toxicity by ER stress proteins and ER stress-inducing drugs.

The present studies examined the toxic interaction between the non-coxib celecoxib derivative OSU-03012 and phosphodiesterase 5 (PDE5) inhibitors, and also determined the roles of endoplasmic reticulum stress response regulators in cell survival. PDE5 inhibitors interacted in a greater than additive fashion with OSU-03012 to kill parental glioma and stem-like glioma cells. Knockdown of the endoplasmic reticulum stress response proteins IRE1 or XBP1 enhanced the lethality of OSU-03012, and of [OSU-03012 + PDE5 inhibitor] treatment. Pan-caspase and caspase-9 inhibition did not alter OSU-03012 lethality but did abolish enhanced killing in the absence of IRE1 or XBP1. Expression of the mitochondrial protective protein BCL-XL or the caspase-8 inhibitor c-FLIP-s, or knockdown of death receptor CD95 or the death receptor caspase-8 linker protein FADD, suppressed killing by [OSU-03012 + PDE5 inhibitor] treatment. CD95 activation was blocked by the nitric oxide synthase inhibitor L-NAME. Knockdown of the autophagy regulatory proteins Beclin1 or ATG5 protected the cells from OSU-03012 and from [OSU-03012 + PDE5 inhibitor] toxicity. Knockdown of IRE1 enhanced OSU-03012/[OSU-03012 + PDE5 inhibitor]-induced JNK activation, and inhibition of JNK suppressed the elevated killing caused by IRE1 knockdown. Knockdown of CD95 blunted JNK activation. Collectively, our data demonstrate that PDE5 inhibitors recruit death receptor signaling to enhance OSU-03012 toxicity in glioblastoma multiforme (GBM) cells.

Abstract 3031: HMGA2 promotes invasion and stemness in glioblastoma

Abstract Glioblastoma Multiforme (GBM) causes the majority of brain tumor-related deaths in the United States. The high invasiveness of GBM prevents complete surgical resection, allowing tumor recurrence. The lack of curative treatment highlights a dire need to develop specific targeted therapeutics. Identifying and targeting novel molecular markers in glioma stem-like cells (GSCs), one of the major causes of tumor invasion and recurrence, could lead to development of better therapies. HMGA2, a non-histone transcriptional modulator, is a regulator of normal and cancer stem cells. The significance of HMGA2 as a potential therapeutic target in GBM has not been investigated. To elucidate the role of HMGA2 in GBM, we have used a combination of in vitro and in vivo techniques. We found increased HMGA2 expression in the mesenchymal subgroup of GBM tumors in the The Cancer Genome Atlas (TCGA) database and in a subset of primary human GBM tumors. HMGA2 is expressed in multiple patient-derived GBM neurosphere lines with increased expression in CD133+ neurospheres compared to CD133- neurospheres. Lentiviral short hairpin RNA (shRNA)-mediated reduction of HMGA2 significantly inhibited invasion of GBM neurosphere lines (040821, 62% reduction with HMGA2 shRNA vs. control shRNA, P<0.001) in transwell invasion assays. Conversely, lentiviral-mediated overexpression of HMGA2 in GBM neurosphere lines increased invasion (GBM1 and 040821, 100-130% increase with HMGA2 overexpression vs. control vector, P<0.001) in transwell invasion assays. In addition, overexpression of HMGA2 increased clonogenicity of GBM neurosphere lines in vitro in soft agar and methylcellulose colony formation assays (40-60% increase with HMGA2 overexpression vs. control vector, P<0.001). Importantly, log-rank analysis of Kaplan-Meier plots showed increased survival of mice injected with GBM neurosphere lines transduced with HMGA2 shRNA compared to control shRNA (P<0.05) in an in vivo, orthotopic, intracranial mouse xenograft model. To elucidate the molecular mechanism by which HMGA2 promotes invasion and stemness in GBM, we performed mRNA expression profiling on GBM neurosphere lines lentivirally transduced to express HMGA2 or empty vector control. We found increased CD44 expression in HMGA2-overexpressing GBM neurosphere lines compared to control. Together, our results suggest an oncogenic role of HMGA2 in promoting GBM tumorigenicity by increasing invasiveness and a stem cell-like state, thereby suggesting that HMGA2 is a potential therapeutic target in GBM. Citation Format: Harpreet Kaur, Marianne Hütt, Xing-gang Mao, Brent A. Orr, Charles G. Eberhart, Eric H. Raabe. HMGA2 promotes invasion and stemness in glioblastoma. [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 3031. doi:10.1158/1538-7445.AM2014-3031

Abstract 4067: TRPM7 channels regulate glioma stem cell through STAT3 and Notch signaling pathways

Abstract A small fraction of cancer stem cells (CSC) initiate and maintain tumors thus driving glioma tumorigenesis and are responsible for resistance to classical chemo- and radio-therapies. It is desirable to identify signaling pathways related to CSC to develop novel therapies to selectively target them. TRPM7 is a ubiquitous Ca2+ and Mg2+ permeable ion channel which contains a serine/threonine kinase. Recent studies have demonstrated that TRPM7 plays an important role in the growth and proliferation of various tumor cells. To determine the possibility of an interaction among TRPM7, Notch, STAT3 and the ALDH1 signaling in the context of brain tumors, we investigated the effects of TRPM7 pathway activation on glioma cells and stem-like cells derived from them. Methods: 1) We analyzed human stem cell signaling pathways using real time RT2 Profile PCR arrays in the A172 glioma cells upon TRPM7 gene silencing. 2) We then examined Notch and STAT3 pathway activation, Aldehyde dehydrogenase 2 family, mitochondrial (ALDH1) and CD133 expressions in parental and glioma stem cell (GSC) populations using quantitative real time-PCR, Western blot, and immunofluorence images. 3) We conducted a ChIP analysis to examine the effects of STAT3 on ALDH1 promoter by introducing constitutive STAT3 (caSTAT3) and dominant STAT3 (dnSTAT3) into A172 cells. 4) Finally, we examined the role of TRPM7 in glioma cell migration and invasion using transwell invasion assay and wound healing assay. Results: 1) RT2 Profile PCR arrays showed that TRPM7 gene silencing down-regulated genes (≥2 fold-change) involved in Notch pathway [Notch1, Jagged 1(JAG1) and Delta-like 1, Drosophila (DLL1)], Wnt pathway, Wingless-type MMTV integration site family, member 1(WNT1) and ALDH1. 2) STAT3 activation and Notch1 expression were decreased in response to TRPM7 silencing. 3) Notch-regulated genes including Notch1, Hey2, JAG1, and Survivin as well as ALDH1 were significantly expressed higher in spheroid glioma CSCs when compared with monolayer cultures. 4) ALDH1 promoter activity was activated by STAT3 in A172 cells. 5) Knockdown of TRPM7 inhibited migration and invasion of A172 cells. Conclusions: TRPM7 activates JAK2/STAT3 and/or Notch signaling pathways and leads to increased cell proliferation and migration. These findings for the first time demonstrate that TRPM7 (1) activates a previously unrecognized STAT3→ALDH1 pathway, and (2) promotes the induction of ALDH1 activity in glioma cells. (This work was supported by the National Institutes of Health grant numbers NIH R01NS047506, R01NS066027, UL1 RR025008, U54 RR026137, AHA 0840132N, and ALZ IIRG-10-173350.) Citation Format: Mingli Liu, Koichi Inoue, Tiandong Leng, Shanchun Guo, Zhigang Xiong. TRPM7 channels regulate glioma stem cell through STAT3 and Notch signaling pathways. [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 4067. doi:10.1158/1538-7445.AM2014-4067

Abstract 1809: Marizomib (NPI-0052) activity as a single agent in malignant glioma

Abstract Background: Glioblastoma multiforme (GBM) is a highly aggressive brain tumor, which displays innate resistance to multiple treatment modalities. Previous studies have shown that the proteasome plays a vital role in the physiology of GBM, and that proteasome inhibition can be used as a strategy for treating malignant gliomas. Marizomib (NPI-0052) is a second generation irreversible proteasome inhibitor, which has a more lipophilic structure and has a broader and more prolonged inhibition profile for 20S proteasome activities compared to bortezomib and carfilzomib, two proteasome inhibitors approved by FDA for treatment of multiple myeloma. While bortezomib and carfilzomib have only modest activity as a treatment of malignant gliomas, marizomib might potentially be a novel therapeutic strategy for primary brain tumors. Unfortunately, to date, the number of studies that have analyzed the effect of marizomib on glioma is limited. Methods: In these studies, we investigated the in vitro activities of marizomib in primary cell cultures derived from a multitude of human brain tumors (high-grade and low-grade gliomas and meningiomas), normal neural stem/progenitor cells (NSC) and as well as in the established human malignant glioma lines U251-MG and D54-MG. The effect of marizomib on cell proliferation, proteasome activity, motility, apoptosis and Reactive Oxygen Species (ROS) were evaluated in glioma cell lines. The inhibition of marizomib by the ROS quenching agent, N-acetyl cysteine (NAC) was also tested. Results: The sensitivities varied in function of the pathology of the tumor, with the malignant glioma stem-like cells being the most severely affected, in contrast with the low-grade glioma, meningioma and NSC-derived cultures. Marizomib inhibited the proliferation of U251-MG and D54-MG cell lines with a half maximal effective concentration (EC50) of 52nM and 20nM respectively, along with a significant decrease in cell migration and invasion. Treatment with marizomib at a concentration of 60nM for 4 hours inhibited proteasome chymotrypsin-like (CT-L, β5) activity by 85% in U251-MG and D54-MG cells. Marizomib treatment of human glioma cells was associated with increased free radical production and apoptosis, along with activation of caspase-3 and cleavage of PARP. Those effects of marizomib can be suppressed by exposure to the ROS quenching agent N-acetyl cysteine (NAC). Conclusion: These preclinical studies demonstrate a significant anti-tumor effect of marizomib in malignant glioma cells. Marizomib has relatively little effect on neural stem/progenitor cells suggesting minimal neurotoxicity, while severely affecting both malignant glioma stem cells and glioma cell lines. But importantly, unlike bortezomib and carfilzomib, marizomib can cross the blood brain barrier. Additional research into the use of marizomib as a potential treatment for malignant gliomas as a single agent or in combination with SOC therapies for glioma is warranted. Citation Format: Kaijun Di, Xing Gong, Dana M. Curticiu, Michael A. Palladino, Daniela A. Bota. Marizomib (NPI-0052) activity as a single agent in malignant glioma. [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 1809. doi:10.1158/1538-7445.AM2014-1809

Abstract 3041: GPR56 promotes the adhesion of glioma stem-like cells to the perivascular niche and regulates cell fate

Abstract INTRODUCTION Gliobastomas (GBM) are the most aggressive and frequent human malignancies in the central nervous system. A small population of cells within the bulk of these tumors displays properties of normal neural stem cells (NSC). Accordingly, these cells are referred to as glioma stem-like initiating cells (GIC). Thus, deregulation of mechanisms that control NSC biology might contribute to the pathogenesis of GBM. NSC and GIC are localized to the perivascular space where they reside in close apposition to blood vessels (BV), which promote self-renewal and keep these cells in an undifferentiated state. GPR56 is an adhesion G-protein coupled receptor expressed in neural progenitors in the embryonic and adult brain and plays an indispensable role in cortical development. Knockout mice studies show that GPR56 mediates the attachment of radial glial endfeet to pial basement membrane (BM). Some of the components of the pial BM are also present in the extracellular matrix surrounding BV, such as laminin and collagen. Therefore, we hypothesized that GPR56 might mediate NSC/GIC localization to the perivascular niche thus contributing to their stem-related properties. EXPERIMENTAL PROCEDURES Here, we have compared the expression of GPR56 in neural progenitors versus more differentiated progeny. In addition, we have assessed the role of GPR56 in the neural stem-like properties of GIC, including their differentiation into more mesenchymal cell types such as endothelial cells or pericytes, by gain- and loss-of-function studies. RESULTS We have found that GPR56 is highly expressed in NSC and its expression is downregulated during differentiation. We have also observed that GPR56 is expressed in cells that reside in the perivascular niche. GPR56-knockdown GIC display impaired adhesion to endothelial cells (EC), whereas GPR56 overexpression increases their adhesion to EC. Furthermore, loss of GPR56 increases the ability of GIC to express endothelial/pericyte markers when co-cultured with EC and increases the CD44+ population, suggesting that GPR56 inhibits the acquisition of a mesenchymal-like phenotype. CONCLUSIONS Taken together, our findings indicate that GPR56 mediates the adhesion of GIC to EC. Moreover, GPR56 inhibits the acquisition of mesenchymal features. Therefore, we suggest that GPR56 might mediate the attachment of GIC to the perivascular niche while preserving their neural stem-like properties by preventing the acquisition of a mesenchymal-like phenotype. Citation Format: Marta Moreno, Stefanie Giera, Xianhua Piao, Nuria de la Iglesia. GPR56 promotes the adhesion of glioma stem-like cells to the perivascular niche and regulates cell fate. [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 3041. doi:10.1158/1538-7445.AM2014-3041

Abstract 4864: Tumor promoting role of NF-kappa B in the glioma environment

Abstract Introduction: Gliomas are the most common primary malignant brain tumors. Their characteristic features are extensive growth and lack of the clear borders due to diffuse invasion of surrounding tissue. Invasive glioma cells have been shown to interact through soluble signals with other cells in the neural microenvironment, which is normally highly inhibitory to cell motility. This communication changes the behavior of the tumor microenvironment to become permissive and facilitate tumor invasion. Here, we investigated the role of NFκB signaling in the glioma microenvironment and demonstrate that activation of NFκB in neural cells is critical for tumor growth and invasion. Methodology: We analyzed the growth and invasion of malignant gliomas using an NFκB-deficient mouse model (RelA(p65)-/- TNFR1-/-). Since p65-/- mice do not survive to adulthood, mouse glioma cells were implanted in p65+/- mice (which have significantly reduced NFκB activity) or their control littermates. Organotypic cultures of p65-/- brain slices and cultures of p65-/- neonatal astrocytes were used to assess the role of active NFκB signaling in astroglia on tumor invasion. Results: Using the mouse glioma cell line GL261 (poorly invasive) we observed significantly reduced tumor volume in NFκB -deficient mice compared to wild-type littermates. Further studies using mouse glioma stem-like cells Mut4 (highly invasive; GFAP-Cre: Nf1loxP/+ Trp53-/+ PTENloxP/+; Alcantara-Llaguno et al., Cancer Cell, 2009) demonstrated reduction both in tumor size and extent of invasion in the NFκB-deficient microenvironment. Importantly, immunohistochemical analysis demonstrated significantly reduced number of protoplasmic GFAP- positive astrocytes surrounding the tumors in p65+/- brains. Further analysis in brain slice cultures showed that invasion of glioblastoma cells was highly reduced through p65+/- tissue. Moreover, p65+/- astrocytes exhibited significantly reduced responses towards glioma cells, including reduced invasion through Matrigel in response to signals released by GL261 cells. Conclusions: Our results strongly suggest that NFκB signaling may be involved in the recruitment or activation of reactive astrocytes surrounding the invasive borders of gliomas. This activation is likely necessary for enhanced tumor invasion through brain tissue. Our results highlight the relevance of the astroglial compartment as a collateral target that should be considered in future strategies targeting brain tumor invasion. Citation Format: Aneta Kwiatkowska, Mohan Sobana Nandhu, Mariano Sebastian Viapiano. Tumor promoting role of NF-kappa B in the glioma environment. [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 4864. doi:10.1158/1538-7445.AM2014-4864

TLR9 is critical for glioma stem cell maintenance and targeting.

Understanding supports for cancer stem-like cells in malignant glioma may suggest therapeutic strategies for their elimination. Here, we show that the Toll-like receptor TLR9 is elevated in glioma stem-like cells (GSC) in which it contributes to glioma growth. TLR9 overexpression is regulated by STAT3, which is required for GSC maintenance. Stimulation of TLR9 with a CpG ligand (CpG ODN) promoted GSC growth, whereas silencing TLR9 expression abrogated GSC development. CpG-ODN treatment induced Frizzled4-dependent activation of JAK2, thereby activating STAT3. Targeted delivery of siRNA into GSC was achieved via TLR9 using CpG-siRNA conjugates. Through local or systemic treatment, administration of CpG-Stat3 siRNA to silence STAT3 in vivo reduced GSC along with glioma growth. Our findings identify TLR9 as a functional marker for GSC and a target for the delivery of efficacious therapeutics for glioma treatment. Cancer Res; 74(18); 5218-28. ©2014 AACR.

P03.08 * IMPROVED YIELD OF STEM-LIKE CELLS USING GLIOBLASTOMA SUSPENSIONS FROM CAVITRON ULTRASONIC SURGERY ASPIRATOR (CUSA) BAGS AS STARTING MATERIAL

Absence of serum and presence of growth factors (mostly EGF and bFGF) has been used to culture cancer stem cells (CSC). In case of GB the term neurospheres (NS) has been considered as synonymous of glioma stem-like cells (GSCs). In our laboratories we isolate and culture GSC from fresh GB specimens growing in the absence of serum and in the presence of EGF and bFGF as neurospheres after mechanical and enzymatic dissociation. Previous data and our experience suggest that NS may mirror more closely than previous, serum-based glioma cell lines, the actual biology of GB. Tumor fragments were used to start the culture. The Cavitation Ultrasonics Surgical Aspirator (CUSA) is increasingly used for GB surgery, decreasing the availability of tumor fragments. CUSA delivers an irrigating solution that converts the fragmented tissue into an emulsion and then aspirates the particles directly into a sterile bag. The bag contains some larger pieces of tissue, debris and high amounts of erythrocytes, and occasionally necrotic or reactive tissue. Here we describe our experience with CUSA bags as the staring material for GSC preparation. Tissue fragments in CUSA bags after several rounds of spinning ad washing are dissociated using the GentleMacs Dissociator (Miltenyi Biotec) that provides a closed system and reproducible results. We have optimized a gentle and effective protocol starting from appropriate gentleMACS programs, allowing to obtain a high yield of viable tumor cells. After processing, cell suspensions are cultured as neurospheres in DMEM/F12, B27 supplement, human recombinant b-FGF and EGF. During the last year we obtained from the Department of Neurosurgery of Istituto Besta a total of 54 CUSA. Primary GB were the most represented brain tumors (87%). Using tumor fragments obtained from surgery and combining mechanical dissociation with enzymatic disaggregation on a series of primary GB we previously obtained GSC in 52% of the cases. This percentage is now increased to 71% with the use of surgical material from CUSA and the optimized protocol. Proliferation kinetics was studied by plating three primary cell lines obtained in parallel from GB specimen and from CUSA material at density of 15,000-30,000 cells/cm2. Cultures were collected every 5 days and the total number of viable cells assessed at each passage by Trypan Blue exclusion. A long term proliferation of cell lines at low sub-culturing stages (3 to 10 passages), showed that proliferation increased exponentially, but the proliferation index of NS from CUSA materials was higher that cells from specimens (1.32 vs 1.18 respectively, p = 0.03). These improvements and the use of a closed system providing a wider margin of safety support the incorporation of this process in a clinical trial protocol which plans to use GSC as a source of antigens for dendritic cell loading, replacing the whole lysate from GB specimens used in current immunotherapy protocols.

P04.13 * EXPRESSION OF PLURIPOTENCY STEMNESS GENES IN HUMAN GLIOMAS OF DIFFERENT TUMOR GRADES BEFORE AND AFTER CHEMOTHERAPY

OBJECTIVE: Following the concept of glioma stem-like cells being the crucial reservoir for self-renewal, differentiation and proliferation of malignant brain tumors and escaping from adjuvant therapy numerous efforts have been made to exactly characterize them. However, several functional assays and analysis of surface markers to clearly identify this subgroup have led to insufficient and controversial results so far. Therefore we analyzed the expression levels of a bunch of potential markers of glioma stem-like cells in human glioma samples of different WHO grades and different treatment. We aimed not only to identify an adequate specific marker for stemness in human gliomas but also to gain insight into expression changes with increasing tumor grade and effects of chemotherapy on cancer stem cell activity. METHODS: cDNA was synthesized from mRNA of 80 snap frozen tumor samples from patients with gliomas of different tumor grades and pre-treatment (WHO grade II, III, secondary glioblastoma ± chemotherapy, primary glioblastoma ± chemotherapy, peritumoral tissue) and from 5 cell lines (U87, Gl36, HES2 (human embryonic stem cells), NPC (neuronal progenitor cells), hDF (human dermal fibroblasts)). Using quantitative real-time PCR relative expression levels of cMyc, Klf4, Nanog, Nestin, Oct4, and Sox2 were analyzed. Quantitative data were normalized in relation to the housekeeping gene SDHA. Results are depicted in mean ± SEM. RESULTS: The expression of Sox2 in all tested tumor groups was higher than in the control peritumoral tissue (2.10 ± 0.72). Interestingly highest expression levels were seen in WHO grade II (8.37 ± 1.60) and grade III (9.80 ± 2.20) tumor groups. cMyc, Nanog and Nestin expression were generally low compared to other tested genes in all tumor samples including control tissue, without any significant differences between the tumor grades and pre-treatment. Oct4 expression was high in all tested tumor groups (mean ranging from 7.5-57.77) compared to other genes. Remarkably Klf4 was lower expressed in two of the tested tumor groups compared to control tissue. CONCLUSIONS: We here provide expression profiles of different stemness genes in human tumors of different grades and treatment on genetic level. Interestingly, the transcription factor Sox2 seems to play a role especially in gliomagenesis as it is overexpressed in lower grade gliomas. At present we are completing our findings with biochemical methods (Western blot analysis).

EGCG inhibits properties of glioma stem-like cells and synergizes with temozolomide through downregulation of P-glycoprotein inhibition.

Combination therapy to inhibit cancer stem cells may have important clinical implications. Here, we examine the molecular mechanisms by which epigallocatechin gallate (EGCG), a bioactive polyphenol in green tea, inhibits the stem cell characteristics of glioma stem-like cells (GSLCs) and synergizes with temozolomide (TMZ), a DNA-methylating agent commonly used as first-line chemotherapy in gliomas. GSLCs were enriched from the human glioblastoma cell line U87 using neurosphere culture. Cells were analyzed using flow cytometry, quantitative PCR, and western blotting. Compared to U87 cells, a higher percentage of U87 GSLCs remained in the G0/G1 phase, with downregulation of the cell-cycle protein CylinD1 and overexpression of stem cell markers CD133 and ALDH1. The drug-resistance gene ABCB1 (but not ABCG2 or MGMT) also showed high mRNA and protein expression. The resistance index of U87 GSLCs against TMZ and carmustine (BCNU) was 3.0 and 16.8, respectively. These results indicate that U87 GSLCs possess neural stem cell and drug-resistance properties. Interestingly, EGCG treatment inhibited cell viability, neurosphere formation, and migration in this cell model. EGCG also induced apoptosis, downregulation of p-Akt and Bcl-2, and cleaving PARP in a dose-dependent manner. Importantly, EGCG treatment significantly downregulated P-glycoprotein expression but not that of ABCG2 or MGMT and simultaneously enhanced sensitivity to TMZ. Our study demonstrates that the use of EGCG alone or in combination with TMZ may be an effective therapeutic strategy for glioma.

Responsiveness of stem-like human glioma cells to all-trans retinoic acid and requirement of retinoic acid receptor isotypes α, β and γ

Retinoic acid (RA) is required for development and homeostasis of the normal mammalian brain and may play a role in the initiation and progression of malignant brain tumors, such as the glioblastoma multiforme (GBM) and the gliosarcoma (Gsarc). The subpopulation of stem-like glioma cells (SLGCs) was shown to resist standard glioma radio-/chemotherapy and to propagate tumor regrowth. We used phenotypically distinct, self-renewing SLGC lines from six human GBMs, two Gsarcs, and two subcloned SLGC derivatives in order to investigate their responsiveness to all-trans retinoic acid (atRA) and to identify the RA-receptor (RAR) isotypes involved. In general, atRA exerted a pro-proliferative and pro-survival effect on SLGCs, though the efficacy was distinct. By means of RAR isotype-selective retinoids we disclosed that these effects were mediated by RARα and RARγ, except for one SLGC line, in which the pro-proliferative signal was induced by the RARβ-selective retinoid. Only one GBM-derived cell line (T1338) and a subpopulation of another (T1389) displayed neural differentiation in response to atRA. Differentiation of T1338 was induced by RARα and RARγ isotype-selective retinoids, associated with down-regulation of Sox2, and the failure to induce orthotopic tumors in the brains of SCID mice. The differential responsiveness of the SLGC lines appeared unrelated to the expression of RARβ, as (i) atRA augmented RAR isotype mRNA expression and particularly rarβ mRNA in all SLGC lines, (ii) rarβ promoter hypomethylation in the SLGC lines was not related to differentiation and (iii) the induction of T1338 differentiation was by RARα- and RARγ-selective ligands.

Therapeutic targeting of malignant glioma.

Glioblastoma Multiforme (GMB) is the most aggressive primary brain tumor with poor survival rates and universal recurrence despite aggressive treatments. Recent research suggested that GBM has multiple glioma cell populations, some of which are organized in a stem cell hierarchical order with different stages of differention. Evidence indicated that recurrence is due to a development or persistance of a subpopulation of these tumor cells which are inherently resistant to treatment and these were defined as the glioma stem-like cells (GSC). It is hypothesized that GSC become highly malignant by accumulating mutations in oncogenic pathways. These cells present with specific surface markers which helps identify them. Targeting the surface markers as well as the signaling pathways of GSCs has been an ongoing research effort. This review focuses on summarizing the current treatment modalities used to glioblastoma treatments, evaluating their efficacy in controlling and eradicatig the GSCs, discussing the machanisms involved in GSC tumor proliferation and resistance to treatments in addition to proposing potential avenues to target GSCs in order to provide a potential cure for this cancer.

Targeting glioma stem-like cells (GSCs) with CpG-siRNA conjugates

Targeting glioma stem-like cells (GSCs) with CpG-siRNA conjugates

MicroRNA-mediated down-regulation of NKG2D ligands contributes to glioma immune escape.

Malignant gliomas are intrinsic brain tumors with a dismal prognosis. They are well-adapted to hypoxic conditions and poorly immunogenic. NKG2D is one of the major activating receptors of natural killer (NK) cells and binds to several ligands (NKG2DL). Here we evaluated the impact of miRNA on the expression of NKG2DL in glioma cells including stem-like glioma cells. Three of the candidate miRNA predicted to target NKG2DL were expressed in various glioma cell lines as well as in glioblastomas in vivo: miR-20a, miR-93 and miR-106b. LNA inhibitor-mediated miRNA silencing up-regulated cell surface NKG2DL expression, which translated into increased susceptibility to NK cell-mediated lysis. This effect was reversed by neutralizing NKG2D antibodies, confirming that enhanced lysis upon miRNA silencing was mediated through the NKG2D system. Hypoxia, a hallmark of glioblastomas in vivo, down-regulated the expression of NKG2DL on glioma cells, associated with reduced susceptibility to NK cell-mediated lysis. This process, however, was not mediated through any of the examined miRNA. Accordingly, both hypoxia and the expression of miRNA targeting NKG2DL may contribute to the immune evasion of glioma cells at the level of the NKG2D recognition pathway. Targeting miRNA may therefore represent a novel approach to increase the immunogenicity of glioblastoma.

Systemic anticancer neural stem cells in combination with a cardiac glycoside for glioblastoma therapy.

The tumor-tropic properties of neural stem cells (NSCs) have been shown to serve as a novel strategy to deliver therapeutic genes to tumors. Recently, we have reported that the cardiac glycoside lanatoside C (Lan C) sensitizes glioma cells to the anticancer agent tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Here, we engineered an FDA-approved human NSC line to synthesize and secrete TRAIL and the Gaussia luciferase (Gluc) blood reporter. We showed that upon systemic injection, these cells selectively migrate toward tumors in the mice brain across the blood-brain barrier, target invasive glioma stem-like cells, and induce tumor regression when combined with Lan C. Gluc blood assay revealed that 30% of NSCs survived 1 day postsystemic injection and around 0.5% of these cells remained viable after 5 weeks in glioma-bearing mice. This study demonstrates the potential of systemic injection of NSCs to deliver anticancer agents, such as TRAIL, which yields glioma regression when combined with Lan C.

Knockdown of nuclear factor erythroid 2-related factor 2 by lentivirus induces differentiation of glioma stem-like cells.

Glioma stem cells (GSCs) are key in the progression and recurrence of glioblastoma. Inducing the differentiation of GSCs is an important therapeutic target for glioblastoma. Nuclear factor erythroid 2-related factor 2 (Nrf2) has been reported to be important in maintaining the stem cell status of GSCs; however, its association with differentiation has not been studied. Herein, we knocked down Nrf2 from GSCs to investigate the role of Nrf2 in the differentiation of GSCs. First, Nrf2 expression was observed at different stages of differentiation; then, Nrf2 was knocked down and the association of Nrf2 with differentiation degree was observed invitro. Finally, GSCs were planted in nude mice to study the association of Nrf2 with differentiation invivo. The expression of Nrf2 decreased with the differentiation process. Following Nrf2 knockdown, the proportion of sphere-like colonies decreased and the dendritic cells in spheres increased; the expression of Nrf2 significantly decreased while the expression of differentiation marker glial fibrillary acidic protein (GFAP) and βIII-tubulin increased both at the protein and the gene level. In the xenografts of nude mice, the differentiation of tumor cells was improved. These results suggest that Nrf2 is a key factor inhibiting the differentiation of GSCs, and knockdown of Nrf2 may promote the differentiation process, providing a therapy target for GSCs.

Autocrine/paracrine sphingosine‐1‐phosphate fuels proliferative and stemness qualities of glioblastoma stem cells

Accumulating reports suggest that human glioblastoma contains glioma stem-like cells (GSCs) which act as key determinants driving tumor growth, angiogenesis, and contributing to therapeutic resistance. The proliferative signals involved in GSC proliferation and progression remain unclear. Using GSC lines derived from human glioblastoma specimens with different proliferative index and stemness marker expression, we assessed the hypothesis that sphingosine-1-phosphate (S1P) affects the proliferative and stemness properties of GSCs. The results of metabolic studies demonstrated that GSCs rapidly consume newly synthesized ceramide, and export S1P in the extracellular environment, both processes being enhanced in the cells exhibiting high proliferative index and stemness markers. Extracellular S1P levels reached nM concentrations in response to increased extracellular sphingosine. In addition, the presence of EGF and bFGF potentiated the constitutive capacity of GSCs to rapidly secrete newly synthesized S1P, suggesting that cooperation between S1P and these growth factors is of central importance in the maintenance and proliferation of GSCs. We also report for the first time that S1P is able to act as a proliferative and pro-stemness autocrine factor for GSCs, promoting both their cell cycle progression and stemness phenotypic profile. These results suggest for the first time that the GSC population is critically modulated by microenvironmental S1P, this bioactive lipid acting as an autocrine signal to maintain a pro-stemness environment and favoring GSC proliferation, survival and stem properties.

EFFECT OF TEMOZOMIDE ON HUMAN DENDRITIC CELLS

BACKGROUND: We are undertaking dendritic cell (DC) therapy against glioma stem-like cells (GSCs) with concomitant maintenance chemotherapy using temozolomide (TMZ). In general, anti-cancer agents are known to suppress immune response in patients. TMZ also suppresses immunity by inducing lymphopenia and myelosuppression in malignant glioma patients. However, there has been no study about the effect of TMZ on DC function. In this study, we evaluate whether concomitant TMZ affects DC function in vitro. METHODS: We isolated human blood plasmacytoid DCs (pDCs) and myelocytoid DCs (mDCs) from healthy volunteers by flow cytometry. These DC subsets were cultured in flat-bottomed 96-well plates at 5 × 104 cells in the final 200 µl of medium per well for 24 h. In the first set of experiment, we assessed cytotoxic activity of TMZ on DCs at clinically relevant concentration. After 24 h, viable cells were measured by a trypan-blue exclusion test. We next measured the Toll-like receptor ligand (TLRL) -induced cytokine production by DCs in the presence of TMZ. DC subsets were stained with FITC-labeled anti-CD83 and PE-labeled anti-CD86 antibody and then analyzed by FACS calibur. We also measured secreted IL-12p40 and IFN-alpha concentration in the supernatant of the cultures by ELISA. RESULTS: Analysis of trypan-blue exclusion of the dead cells showed that over 300 microM of TMZ started to kill both DC subsets and there was no cytocidal effect at 100 microM, a clinically relevant concentration. Therefore, we thereafter used 10 microM to 300 microM for the following assays. We found that TMZ did not affect IFN-alpha production by pDCs and IL-12 production by mDCs in response to various TLRLs. In addition, up to 100 microM of concentration, TMZ did not influence the CD83 and CD86 expression on both DC subsets. CONCLUSIONS: Temozolomide did not significantly affect on DC function, suggesting that DC therapy and concomitant chemotherapy using TMZ may be possible, although further assessment in the clinical setting is necessary. In the presentation, we also would like to present preliminary data on interaction between DCs and two types of GSCs (proneural and mesenchymal). SECONDARY CATEGORY: n/a.

"GO OR GROW" - LINKS BETWEEN CELLULAR FUNCTION, GLUCOSE METABOLISM AND GLIOMA MICROENVIRONMENT

BACKGROUND: The go or grow hypothesis states that glioma cells either favor proliferation at the expense of migration or migration at the expense of proliferation. This dichotomy is regulated by multiple factors, including ECM components, microRNAs and transcription factors. We recently found that glucose metabolism also contributes to this dichotomy and discovered an oxygen-dependent metabolic switch between the pentose phosphate pathway (PPP) and the preparatory phase of glycolysis in glioma stem-like cells and tissues, which is associated with distinct phenotypes. Expression of glycolysis enzymes and glycolytic flux are increased by hypoxia and associated with cell migration, whereas the PPP is favored by oxygenation and associated with proliferation (Kathagen et al., Acta Neuropathol 2013). Building on these findings, we now studied the causal regulatory involvement of glycolysis versus the PPP in migration versus proliferation. METHODS: Enzyme inhibitors and lentiviral shRNA transduction were used to modulate the expression and activity of enzymes of glycolysis and the PPP. Proliferation, migration and clonogenicity were analyzed in vitro. Tumor initiation and growth were studied in an orthotopic nude mouse model. RESULTS: We identified G6PD and ALDOC as the most strongly inversely regulated enzymes of the PPP and glycolysis, respectively. Stable knockdown of G6PD, the first and key regulatory PPP enzyme, resulted in strikingly reduced proliferation but increased migration of GS cells. Conversely, knockdown of ALDOC inhibited migration while favoring proliferation. Similar results were obtained using enzyme inhibitors. The G6PD inhibitor 6-aminonicotinamide inhibited proliferation while stimulating migration, whereas erythrose-4-phosphate, an inhibitor of the glycolysis enzyme glucose-6-phosphate-isomerase (GPI) decreased migration. 2-deoxyglucose, a hexokinase inhibitor inhibited both proliferation and migration. Knockdown of G6PD in a highly proliferative but non-invasive conventional serum-grown glioblastoma cell line resulted in prolonged survival in vivo, whereas knockdown of ALDOC shortened survival. Interestingly, GPI is also known as a secreted factor termed autocrine motility factor (AMF), which binds to the surface receptor AMFR. Hypoxia strongly increased the secretion of GPI/AMF from GS cells as well as the expression of AMFR. Recombinant human GPI/AMF stimulated the migration of GS cells even more strongly than hepatocyte growth factor, a known potent glioma cell motogen. CONCLUSIONS: By interference with key enzymes of glycolysis and the PPP we demonstrated a direct causal relevance of the two pathways for the go or grow behaviour of GS cells, respectively. The identification of GPI/AMF as an extraordinarily potent motogen for GS cells, explains in part the pro-migratory effect of glycolysis. SECONDARY CATEGORY: Neuropathology & Tumor Biomarkers.

PI3K and Bcl-2 inhibition primes glioblastoma cells to apoptosis through downregulation of Mcl-1 and Phospho-BAD.

Glioblastoma multiforme (GBM) is a highly malignant human brain neoplasm with limited therapeutic options. GBMs display a deregulated apoptotic pathway with high levels of the antiapoptotic Bcl-2 family of proteins and overt activity of the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Therefore, combined interference of the PI3K pathway and the Bcl-2 family of proteins is a reasonable therapeutic strategy. ABT-263 (Navitoclax), an orally available small-molecule Bcl-2 inhibitor, and GDC-0941, a PI3K inhibitor, were used to treat established glioblastoma and glioblastoma neurosphere cells, alone or in combination. Although GDC-0941 alone had a modest effect on cell viability, treatment with ABT-263 displayed a marked reduction of cell viability and induction of apoptotic cell death. Moreover, combinatorial therapy using ABT-263 and GDC-0941 showed an enhanced effect, with a further decrease in cellular viability. Furthermore, combination treatment abrogated the ability of stem cell-like glioma cells to form neurospheres. ABT-263 and GDC-0941, in combination, resulted in a consistent and significant increase of Annexin V positive cells and loss of mitochondrial membrane potential compared with either monotherapy. The combination treatment led to enhanced cleavage of both initiator and effector caspases. Mechanistically, GDC-0941 depleted pAKT (Serine 473) levels and suppressed Mcl-1 protein levels, lowering the threshold for the cytotoxic actions of ABT-263. GDC-0941 decreased Mcl-1 in a posttranslational manner and significantly decreased the half-life of Mcl-1 protein. Ectopic expression of human Mcl-1 mitigated apoptotic cell death induced by the drug combination. Furthermore, GDC-0941 modulated the phosphorylation status of BAD, thereby further enhancing ABT-263-mediated cell death. Combination therapy with ABT-263 and GDC-0941 has novel therapeutic potential by specifically targeting aberrantly active, deregulated pathways in GBM, overcoming endogenous resistance to apoptosis.