Proliferation Of Glioblastoma Stem-like Cells Research Articles

CNSC-09. THE NEUROENDOCRINE FACTOR VGF IS NECESSARY FOR THE MAINTENANCE OF THE TUMOR STEM CELL POPULATION IN GLIOBLASTOMA

Abstract A major factor that contributes to the unchecked growth and recurrence of glioblastoma (GBM) tumors is the presence of a heterogeneous population of GBM stem-like cells (GSCs) that continuously replenish the tumor mass. Targeting strategies against this key cell population have rarely considered the trophic signals from peritumoral neural cells, which can protect the tumor cells during therapy and therefore promote recurrence. Here, we have focused on the little-known secreted factor VGF and its derived neuropeptides, which are largely restricted to the CNS and mediate paracrine communication between neural cells. We analyzed over 200 tissue specimens and cultured GBM cells, demonstrating that VGF is upregulated in malignant gliomas and expressed in GSCs of different subtypes, although it is markedly reduced in differentiated GBM cells. More importantly, we detected VGF expression in peritumoral astrocytes, even though this neuroendocrine factor has never been described in glial cells in normal brain. Co-culture experiments and scRNAseq data from clinical tumors confirmed that VGF is expressed in tumor-associated astrocytes, suggesting that these cells are a paracrine source of VGF in GBM. Accordingly, purified exogenous VGF increased the proliferation of GSCs and rescued GSC viability when the tumor cells were treated with the chemotherapeutic temozolomide. Moreover, purified VGF decreased the expression of inflammatory cytokines in activated macrophages, suggesting that this factor may also contribute to local immunosuppression in gliomas. Conversely, knockdown of VGF in GSCs was sufficient to decrease their viability, self-renewal, and expression of stemness-related genes. At the molecular level, VGF increased intracellular calcium and Akt/mTOR signaling in GSCs, suggesting a possible G protein coupled receptor-mediated mechanism of action. Taken together, our results explore for the first time the role of brain-derived VGF in GBM progression, highlighting the importance of disrupting the communication between the tumor and its microenvironment to achieve long-term anti-tumor efficacy.

FMRP modulates the Wnt signalling pathway in glioblastoma

Converging evidence indicates that the Fragile X Messenger Ribonucleoprotein (FMRP), which absent or mutated in Fragile X Syndrome (FXS), plays a role in many types of cancers. However, while FMRP roles in brain development and function have been extensively studied, its involvement in the biology of brain tumors remains largely unexplored. Here we show, in human glioblastoma (GBM) biopsies, that increased expression of FMRP directly correlates with a worse patient outcome. In contrast, reductions in FMRP correlate with a diminished tumor growth and proliferation of human GBM stem-like cells (GSCs) in vitro in a cell culture model and in vivo in mouse brain GSC xenografts. Consistently, increased FMRP levels promote GSC proliferation. To characterize the mechanism(s) by which FMRP regulates GSC proliferation, we performed GSC transcriptome analyses in GSCs expressing high levels of FMRP, and in these GSCs after knockdown of FMRP. We show that the WNT signalling is the most significantly enriched among the published FMRP target genes and genes involved in ASD. Consistently, we find that reductions in FMRP downregulate both the canonical WNT/β-Catenin and the non-canonical WNT-ERK1/2 signalling pathways, reducing the stability of several key transcription factors (i.e. β-Catenin, CREB and ETS1) previously implicated in the modulation of malignant features of glioma cells. Our findings support a key role for FMRP in GBM cancer progression, acting via regulation of WNT signalling.

Abstract 3114: Olfactory ensheathing glia as a cell-based therapy for glioblastomas

Abstract Introduction: Olfactory ensheathing glia (OEG) is a fully differentiated cell that promotes the continuous regeneration of odor receptors from the nasal mucosa to the olfactory bulb during adult lifespan. OEG secretes several factors making the environment more permissive to regeneration, and promotes differentitation of progenitor/neural stem cells. Recently, our group showed for the first time that OEG can target glioblastoma (GBM) cells and GBM stem-like cells (GSC) in the mice brain via intranasal delivery, the natural route of OEG to the brain. Here, we test the potential of OEG in inhibiting the proliferation and stemness of GSC, leading to their differentiation (similar to their neural stem cells counterpart), making them susceptible to conventional therapy. Methods: OEG derived from mice OB (1.5e+5 cells) were co-cultured with GSC at 1:1 ratio. We used a dual secreted luciferase reporter that can be multiplexed together to monitor both OEG and GSCs viability and proliferation in the same well. For in vivo analysis, mice-bearing patient-derived xenograft GSCs were treated with a single injection of 1e+5 OEG intranasally or control. One week later, each group was subdivided into two groups which either received ionizing radiation (IR, one dose of 4Gy) or control. Results: GSC or their corresponding differentiated glioma cells (cultured in serum for 10 days) displayed a decrease in cell viability when co-cultured with OEG compared to control. Analysis of mRNA levels and immunofluorescence images from these co-cultures (using a Transwell) showed a significant decrease in markers associated with stemness and aggressiveness, and an increase in both neuronal and astrocytic markers compared to control. When assessed tumor formation, GSC cultured with OEG conditioned media (OEGCM) failed to form tumors and became more susceptible to IR and Temozolomide. In mice bearing PDX, intranasal injection of non-modified OEG lead to inhibition of tumor growth prolonged mouse survival (48 days vs 34 days for control; P<0.0001). IR had a modest effect on this model (38 days; P<0.005 vs control). The combination of a single OEG injection and IR had the most therapeutic effect on tumor growth and animal survival (54 days; P<0.0001 vs control). Conclusion: Our findings show that OEG inhibits GBM stemness and malignancy, induces differentiation of GSCs, making them prone to conventional therapy. OEG have the advantage over other typical cell therapies since they are terminally differentiated and can be easily obtained from the olfactory mucosa, a simple procedure typically done for patients with spinal cord injury allowing autologous transplantation. Citation Format: Litia Carvalho, Renata Fleming, Gulsah Erel-Akbaba, Ghazal Lashgari, Daniel Ryan, Elie Tabet, Max Zinter, Jian Teng, Bakhos Tannous. Olfactory ensheathing glia as a cell-based therapy for glioblastomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3114.

Dual Role of Integrin Alpha-6 in Glioblastoma: Supporting Stemness in Proneural Stem-Like Cells While Inducing Radioresistance in Mesenchymal Stem-Like Cells.

Simple SummaryGlioblastoma stem-like cells (GSCs) are responsible for most of the malignant characteristics of glioblastoma, including therapeutic resistance, tumour recurrence, and tumour cellular heterogeneity. Therefore, increased understanding of the mechanisms regulating GSCs aggressiveness may help to improve patients’ outcomes. Here, we investigated the role of integrin a6 in controlling stemness and resistance to radiotherapy across proneural and mesenchymal molecular subtypes. We observed that integrin a6 had a clear role in stemness maintenance in proneural but not in mesenchymal GSCs. In addition, we proved a crucial role of integrin a6 in supporting mesenchymal GSCs resistance to ionizing radiation. Finally, we highlighted that integrin a6 may control different stem-associated features in GSCs, depending on the molecular subtype. The inhibition of integrin a6 limits stem-like malignant characteristics in both GSCs subtypes and thus may potentially control tumour relapse following conventional treatment.Therapeutic resistance after multimodal therapy is the most relevant cause of glioblastoma (GBM) recurrence. Extensive cellular heterogeneity, mainly driven by the presence of GBM stem-like cells (GSCs), strongly correlates with patients’ prognosis and limited response to therapies. Defining the mechanisms that drive stemness and control responsiveness to therapy in a GSC-specific manner is therefore essential. Here we investigated the role of integrin a6 (ITGA6) in controlling stemness and resistance to radiotherapy in proneural and mesenchymal GSCs subtypes. Using cell sorting, gene silencing, RNA-Seq, and in vitro assays, we verified that ITGA6 expression seems crucial for proliferation and stemness of proneural GSCs, while it appears not to be relevant in mesenchymal GSCs under basal conditions. However, when challenged with a fractionated protocol of radiation therapy, comparable to that used in the clinical setting, mesenchymal GSCs were dependent on integrin a6 for survival. Specifically, GSCs with reduced levels of ITGA6 displayed a clear reduction of DNA damage response and perturbation of cell cycle pathways. These data indicate that ITGA6 inhibition is able to overcome the radioresistance of mesenchymal GSCs, while it reduces proliferation and stemness in proneural GSCs. Therefore, integrin a6 controls crucial characteristics across GBM subtypes in GBM heterogeneous biology and thus may represent a promising target to improve patient outcomes.

MELK Inhibition Effectively Suppresses Growth of Glioblastoma and Cancer Stem-Like Cells by Blocking AKT and FOXM1 Pathways.

Glioblastoma multiforme (GBM) is a devastating disease yet no effective drug treatment has been established to date. Glioblastoma stem-like cells (GSCs) are insensitive to treatment and may be one of the reasons for the relapse of GBM. Maternal embryonic leucine zipper kinase gene (MELK) plays an important role in the malignant proliferation and the maintenance of GSC stemness properties of GBM. However, the therapeutic effect of targeted inhibition of MELK on GBM remains unclear. This study analyzed the effect of a MELK oral inhibitor, OTSSP167, on GBM proliferation and the maintenance of GSC stemness. OTSSP167 significantly inhibited cell proliferation, colony formation, invasion, and migration of GBM. OTSSP167 treatment reduced the expression of cell cycle G2/M phase-related proteins, Cyclin B1 and Cdc2, while up-regulation the expression of p21 and subsequently induced cell cycle arrest at the G2/M phase. OTSSP167 effectively prolonged the survival of tumor-bearing mice and inhibited tumor cell growth in in vivo mouse models. It also reduced protein kinase B (AKT) phosphorylation levels by OTSSP167 treatment, thereby disrupting the proliferation and invasion of GBM cells. Furthermore, OTSSP167 inhibited the proliferation, neurosphere formation and self-renewal capacity of GSCs by reducing forkhead box M1 (FOXM1) phosphorylation and transcriptional activity. Interestingly, the inhibitory effect of OTSSP167 on the proliferation of GSCs was 4-fold more effective than GBM cells. In conclusion, MELK inhibition suppresses the growth of GBM and GSCs by double-blocking AKT and FOXM1 signals. Targeted inhibition of MELK may thus be potentially used as a novel treatment for GBM.

Differentiation of glioblastoma stem cells promoted by miR-128 or miR-302a overexpression enhances senescence-associated cytotoxicity of axitinib.

Despite the intense global efforts towards an effective treatment of glioblastoma (GB), current therapeutic options are unsatisfactory with a median survival time of 12-15months after diagnosis, which has not improved significantly over more than a decade. The high tumoral heterogeneity confers resistance to therapies, which has hindered a successful clinical outcome, GB remaining among the deadliest cancers. A hallmark of GB is its high recurrence rate, which has been attributed to the presence of a small subpopulation of tumor cells called GB stem-like cells (GSC). In the present work, the efficacy of a multimodal strategy combining microRNA (miRNA) modulation with new generation multitargeted tyrosine kinase inhibitors (imatinib and axitinib) was investigated aiming at tackling this subpopulation of GB cells. MiR-128 and miR-302a were selected as attractive therapeutic candidates on the basis of previous findings reporting that reestablishment of their decreased expression levels in GSC resulted in cell differentiation, which could represent a possible strategy to sensitize GSC to chemotherapy. Our results show that overexpression of miR-128 or miR-302a induced GSC differentiation, which enhanced senescence mediated by axitinib treatment, thus further impairing GSC proliferation. We also provided evidence for the capacity of GSC to efficiently internalize functionalized stable nucleic acid lipid particles, previously developed and successfully applied in our laboratory to target GB. Taken together, our findings will be important in the future design of a GB-targeted multimodal miRNA-based gene therapy, combining overexpression of miR-128 or miR-302a with axitinib treatment, endowed with the ability to overcome drug resistance.

Abstract 4956: PrPc depletion in glioblastoma patient derived xenograft cells by CRISPR/Cas9

Abstract Glioblastoma (GBM), the most common primary malignant brain tumor, exhibits a highly proliferative cell subpopulation called Glioblastoma stem-like cells (GSCs), presumed to contribute to the aggressive behavior of GBM. Our group has shown that the Prion Protein (PrPC), a cell surface glycoprotein and regulator of several stem cell functions, is enriched in GSCs and co-expresses with conventional GSCs markers. PrPC has been proposed as a scaffold protein, able to govern the assembly of multi-component complexes in the cell membrane. Integrin α6 (ITGA6), a cell surface laminin receptor, is enriched in GSCs as well, and exhibits relevant effects on GSCs biology, related to self-renewal and migration. To investigate a potential association between PrPC and ITGA6, we will perform loss-of-function experiments in GSCs via siRNA and CRISPR/Cas9 to characterize these cell surface molecules expression and their effect on GSCs proliferation, migration and invasion. Preliminary results show that depletion of PrPC perturbates ITGA6 labeling on plasma membrane of GSC. Thus far, cells from patient-derived xenografts (GBM33-PDX2) were transfected a construct for PrPC knockout by CRISPR/Cas9 via Nucleofector Technology. We found around 60% GFP-positive cells via flow cytometry for the CRISPR pool and we are currently isolating clones to confirm PrPC expression. Remarkably, the colonies exhibited distinct morphologies such as neural-like, epithelial-like and stem cell-like, indicating the heterogenous nature of this tumor. Western blotting assays will be performed to assess PrPC and ITGA6 expression in both commercial cell line U87 and GBM33-PDX2. The characterization of PrPC-ITGA6 association might provide insight into GSCs behavior and new anti-GBM therapeutic strategies. Citation Format: Maria Isabel Melo Escobar, Barbara Paranhos Coelho, Mariana Brandão Prado, Rebeca Iglesia, Marilene Hohmuth Lopes. PrPc depletion in glioblastoma patient derived xenograft cells by CRISPR/Cas9 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4956.

Mir-370-3p Impairs Glioblastoma Stem-Like Cell Malignancy Regulating a Complex Interplay between HMGA2/HIF1A and the Oncogenic Long Non-Coding RNA (lncRNA) NEAT1

Glioblastoma (GBM) is the most aggressive and prevalent form of a human brain tumor in adults. Several data have demonstrated the implication of microRNAs (miRNAs) in tumorigenicity of GBM stem-like cells (GSCs). The regulatory functions of miRNAs in GSCs have emerged as potential therapeutic candidates for glioma treatment. The current study aimed at investigating the function of miR-370-3p in glioma progression, as aberrant expression of miR-370-3p, is involved in various human cancers, including glioma. Analyzing our collection of GBM samples and patient-derived GSC lines, we found the expression of miR-370-3p significantly downregulated compared to normal brain tissues and normal neural stem cells. Restoration of miR-370-3p expression in GSCs significantly decreased proliferation, migration, and clonogenic abilities of GSCs, in vitro, and tumor growth in vivo. Gene expression analysis performed on miR-370-3p transduced GSCs, identified several transcripts involved in Epithelial to Mesenchymal Transition (EMT), and Hypoxia signaling pathways. Among the genes downregulated by the restored expression of miR-370-3p, we found the EMT-inducer high-mobility group AT-hook 2 (HMGA2), the master transcriptional regulator of the adaptive response to hypoxia, Hypoxia-inducible factor (HIF)1A, and the long non-coding RNAs (lncRNAs) Nuclear Enriched Abundant Transcript (NEAT)1. NEAT1 acts as an oncogene in a series of human cancers including gliomas, where it is regulated by the Epidermal Growth Factor Receptor (EGFR) pathways, and contributes to tumor growth and invasion. Noteworthy, the expression levels of miR-370-3p and NEAT1 were inversely related in both GBM tumor specimens and GSCs, and a dual-luciferase reporter assay proved the direct binding between miR-370-3p and the lncRNAs NEAT1. Our results identify a critical role of miR-370-3p in the regulation of GBM development, indicating that miR-370-3p acts as a tumor-suppressor factor inhibiting glioma cell growth, migration and invasion by targeting the lncRNAs NEAT1, HMGA2, and HIF1A, thus, providing a potential candidate for GBM patient treatment.

Curcumin Loaded in Niosomal Nanoparticles Improved the Anti-tumor Effects of Free Curcumin on Glioblastoma Stem-like Cells: an In Vitro Study

Using a novel curcumin-loaded niosome nanoparticle (CM-NP), the present study was designed to evaluate the effect of curcumin on human glioblastoma stem-like cells (GSCs). CM-NP has a diameter of ~ 60 nm and a zeta potential of ~ − 35 mV with a constant physicochemical stability. The cytotoxic effects of free curcumin (CM) and CM-NP were investigated on GSCs obtained during the removal of a brain tumor. Both CM and CM-NP caused a dose-dependent decrease in cell proliferation and viability of GSCs. The IC50 values of CM and CM-NP on GSCs were 50 and 137 μg/ml after 24 h, respectively. CM-NP exerted significantly higher effects on GSC viability, apoptosis, cell cycle arrest, and the expression of Bax, a pro-apoptotic marker, compared with CM. In addition, the migration of GSCs was significantly impaired following the administration of CM-NP compared with CM. Furthermore, CM-NP significantly increased the values of reactive oxygen species and decreased the mRNA expressions of NF-κB and IL-6 of GSCs compared with CM. Our data also revealed that CM-NP could significantly reduce the invasiveness of GSCs compared with CM, possibly via MCP-1-mediated pathways. In addition, CM-NP exhibited a significantly greater inhibitory effect on colony formation of GSCs compared with CM. These data indicate that CM-NP exhibited stronger anti-tumor effects on GSCs than CM. Although further in vivo investigations are warranted, our results suggest that CM-NP could be an ideal carrier to deliver curcumin for potential therapeutic approaches into glioblastoma.

Glioblastoma chemoresistance: roles of the mitochondrial melatonergic pathway.

Treatment-resistance is common in glioblastoma (GBM) and the glioblastoma stem-like cells (GSC) from which they arise. Current treatment options are generally regarded as very poor and this arises from a poor conceptualization of the biological underpinnings of GBM/GSC and of the plasticity that these cells are capable of utilizing in response to different treatments. A number of studies indicate melatonin to have utility in the management of GBM/GSC, both per se and when adjunctive to chemotherapy. Recent work shows melatonin to be produced in mitochondria, with the mitochondrial melatonergic pathway proposed to be a crucial factor in driving the wide array of changes in intra- and inter-cellular processes, as well as receptors that can be evident in the cells of the GBM/GSC microenvironment. Variations in the enzymatic conversion of N-acetylserotonin (NAS) to melatonin may be especially important in GSC, as NAS can activate the tyrosine receptor kinase B to increase GSC survival and proliferation. Consequently, variations in the NAS/melatonin ratio may have contrasting effects on GBM/GSC survival. It is proposed that mitochondrial communication across cell types in the tumour microenvironment is strongly driven by the need to carefully control the mitochondrial melatonergic pathways across cell types, with a number of intra- and inter-cellular processes occurring as a consequence of the need to carefully regulate the NAS/melatonin ratio. This better integrates previously disparate data on GBM/GSC as well as providing clear future research and treatment options.

NF\u03baB activation in differentiating glioblastoma stem-like cells is promoted by hyaluronic acid signaling through TLR4

We have previously described that the NFκB pathway is upregulated during differentiation of glioblastoma stem-like cells (GSCs) which keeps differentiating GSCs in a proliferative astrocytic precursor state. However, extracellular signals and cellular mediators of this pathway are not clear yet. Here, we show that TLR4 is a key factor to promote NFκB activation in differentiating GSCs. TLR4 is upregulated during differentiation of GSCs and promotes transcriptional activation of NFκB as determined by luciferase-reporter assays and expression of NFκB target genes. Downregulation of TLR4 by shRNAs or blockade with anti-TLR4 specific antibodies drastically inhibited NFκB activity which promoted further differentiation and reduced proliferation of GSCs. We found that hyaluronic acid (HA), a main component of brain extracellular matrix, triggers the TLR4-NFκB pathway in differentiating GSCs. Moreover, HA is synthesized and released by GSCs undergoing differentiation and leads to transcriptional activation of NFκB, which is inhibited following downregulation of TLR4 or blockade of HA synthesis. Thus, we have demonstrated that during the process of differentiation, GSCs upregulate TLR4 and release the TLR4 ligand HA, which activates the TLR4-NFκB signaling pathway. This strategy may efficiently be used by differentiating GSCs to maintain their proliferative potential and consequently their tumorigenic capacity.

Modeling Microenvironmental Regulation of Glioblastoma Stem Cells: A Biomaterials Perspective

Following diagnosis of a glioblastoma (GBM) brain tumor, surgical resection, chemotherapy and radiation together yield a median patient survival of only 15 months. Importantly, standard treatments fail to address the dynamic regulation of the brain tumor microenvironment that actively supports tumor progression and treatment resistance. It is becoming increasingly recognized that specialized niches within the tumor microenvironment maintain a population of highly malignant glioblastoma stem-like cells (GSCs). GSCs are resistant to traditional chemotherapy and radiation therapy, suggesting that they may be responsible for the near universal rates of tumor recurrence and associated morbidity in GBM. Thus, disrupting microenvironmental support for GSCs could be critical to developing more effective GBM therapies. Three-dimensional (3D) culture models of the tumor microenvironment are powerful tools for identifying key biochemical and biophysical inputs that impact malignant behaviors. Such systems have been used effectively to identify conditions that regulate GSC proliferation, invasion, stem-specific phenotypes, and treatment resistance. Considering the significant role that GSC microenvironments play in regulating this tumorigenic sub-population, these models may be essential for uncovering mechanisms that limit GSCs malignancy.

Abstract 4996: The m6A hallmark of cancer: RNA demethylase ALKBH5 maintains tumorigenicity of glioblastoma stem-like cells by sustaining FOXM1 expression and cell proliferation

Abstract N6-methyladenosine (m6A) is the most prevalent internal modification on mRNA, but its functions in human diseases are poorly understood. The dynamic and reversible m6A modification installed and erased by N6-methyltransferases and demethylases regulates cell fate and gene expression. We show that the m6A demethylase ALKBH5 is highly expressed in patient-derived glioblastoma stem-like cells (GSCs) and informs poor survival of patients with glioblastoma. Silencing ALKBH5 suppresses the proliferation of GSCs in vitro and in vivo. Results of integrated transcriptome and m6A-seq analyses reveal a global change of gene expression enriched in cell cycle and altered expression of select ALKBH5 targets. Among these targets, Ingenuity’s upstream regulator analysis identified FOXM1, an essential transcription factor for GSC proliferation, as a key mediator responsible for the disrupted proliferation program. Using multiple molecular and biochemical approaches, we demonstrate that ALKBH5 binds to and demethylates FOXM1 nascent transcripts. This promotes the interaction of FOXM1 pre-mRNA with HuR, thereby maintaining FOXM1 expression. Further, a long noncoding RNA antisense to the FOXM1 (FOXM1-AS) interacts with ALKBH5 as well as FOXM1 pre-mRNA and promotes their interaction. Depleting ALKBH5 and FOXM1-AS abolishes GSC tumorigenesis through the FOXM1 axis. The vulnerability of GSCs to disruptions in ALKBH5-dependent gene expression suggests that m6A has a central role in tumor development and provides a rationale for therapeutically targeting epitranscriptomic modulators in patients with glioblastoma. Citation Format: Sicong Zhang, Aidong Zhou, Oliver Bogler, Sadhan Majumder, Suyun Huang. The m6A hallmark of cancer: RNA demethylase ALKBH5 maintains tumorigenicity of glioblastoma stem-like cells by sustaining FOXM1 expression and cell proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4996. doi:10.1158/1538-7445.AM2017-4996

Engagement of cellular prion protein with the co-chaperone Hsp70/90 organizing protein regulates the proliferation of glioblastoma stem-like cells

BackgroundGlioblastoma (GBM), a highly aggressive brain tumor, contains a subpopulation of glioblastoma stem-like cells (GSCs) that play roles in tumor maintenance, invasion, and therapeutic resistance. GSCs are therefore a promising target for GBM treatment. Our group identified the cellular prion protein (PrPC) and its partner, the co-chaperone Hsp70/90 organizing protein (HOP), as potential target candidates due to their role in GBM tumorigenesis and in neural stem cell maintenance.MethodsGSCs expressing different levels of PrPC were cultured as neurospheres with growth factors, and characterized with stem cells markers and adhesion molecules markers through immunofluorescence and flow cytometry. We than evaluated GSC self-renewal and proliferation by clonal density assays and BrdU incorporation, respectively, in front of recombinant HOP treatment, combined or not with a HOP peptide which mimics the PrPC binding site. Stable silencing of HOP was also performed in parental and/or PrPC-depleted cell populations, and proliferation in vitro and tumor growth in vivo were evaluated. Migration assays were performed on laminin-1 pre-coated glass.ResultsWe observed that, when GBM cells are cultured as neurospheres, they express specific stemness markers such as CD133, CD15, Oct4, and SOX2; PrPC is upregulated compared to monolayer culture and co-localizes with CD133. PrPC silencing downregulates the expression of molecules associated with cancer stem cells, upregulates markers of cell differentiation and affects GSC self-renewal, pointing to a pivotal role for PrPC in the maintenance of GSCs. Exogenous HOP treatment increases proliferation and self-renewal of GSCs in a PrPC-dependent manner while HOP knockdown disturbs the proliferation process. In vivo, PrPC and/or HOP knockdown potently inhibits the growth of subcutaneously implanted glioblastoma cells. In addition, disruption of the PrPC-HOP complex by a HOP peptide, which mimics the PrPC binding site, affects GSC self-renewal and proliferation indicating that the HOP-PrPC complex is required for GSC stemness. Furthermore, PrPC-depleted GSCs downregulate cell adhesion-related proteins and impair cell migration indicating a putative role for PrPC in the cell surface stability of cell adhesion molecules and GBM cell invasiveness, respectively.ConclusionsIn conclusion, our results show that the modulation of HOP-PrPC engagement or the decrease of PrPC and HOP expression may represent a potential therapeutic intervention in GBM, regulating glioblastoma stem-like cell self-renewal, proliferation, and migration.

M6A Demethylase ALKBH5 Maintains Tumorigenicity of Glioblastoma Stem-like Cells by Sustaining FOXM1 Expression and Cell Proliferation Program

The dynamic and reversible N6-methyladenosine (m6A) RNA modification installed and erased by N6-methyltransferases and demethylases regulates gene expression and cell fate. We show that the m6A demethylase ALKBH5 is highly expressed in glioblastoma stem-like cells (GSCs). Silencing ALKBH5 suppresses the proliferation of patient-derived GSCs. Integrated transcriptome and m6A-seq analyses revealed altered expression of certain ALKBH5 target genes, including the transcription factor FOXM1. ALKBH5 demethylates FOXM1 nascent transcripts, leading to enhanced FOXM1 expression. Furthermore, a long non-coding RNA antisense to FOXM1 (FOXM1-AS) promotes the interaction of ALKBH5 with FOXM1 nascent transcripts. Depleting ALKBH5 and FOXM1-AS disrupted GSC tumorigenesis through the FOXM1 axis. Our work uncovers a critical function for ALKBH5 and provides insight into critical roles of m6A methylation in glioblastoma.

TRIM8 regulates stemness in glioblastoma through PIAS3‐STAT3

Glioblastoma (GBM) is the most malignant form of primary brain tumor, and GBM stem‐like cells (GSCs) contribute to the rapid growth, therapeutic resistance, and clinical recurrence of these fatal tumors. STAT3 signaling supports the maintenance and proliferation of GSCs, yet regulatory mechanisms are not completely understood. Here, we report that tri‐partite motif‐containing protein 8 (TRIM8) activates STAT3 signaling to maintain stemness and self‐renewing capabilities of GSCs. TRIM8 (also known as ‘glioblastoma‐expressed ring finger protein’) is expressed equally in GBM and normal brain tissues, despite its hemizygous deletion in the large majority of GBMs, and its expression is highly correlated with stem cell markers. Experimental knockdown of TRIM8 reduced GSC self‐renewal and expression of SOX2, NESTIN, and p‐STAT3, and promoted glial differentiation. Overexpression of TRIM8 led to higher expression of p‐STAT3, c‐MYC, SOX2, NESTIN, and CD133, and enhanced GSC self‐renewal. We found that TRIM8 activates STAT3 by suppressing the expression of PIAS3, an inhibitor of STAT3, most likely through E3‐mediated ubiquitination and proteasomal degradation. Interestingly, we also found that STAT3 activation upregulates TRIM8, providing a mechanism for normalized TRIM8 expression in the setting of hemizygous gene deletion. These data demonstrate that bidirectional TRIM8‐STAT3 signaling regulates stemness in GSC.

The effects of tumor treating fields and temozolomide in MGMT expressing and non-expressing patient-derived glioblastoma cells

A recent Phase 3 study of newly diagnosed glioblastoma (GBM) demonstrated the addition of tumor treating fields (TTFields) to temozolomide (TMZ) after combined radiation/TMZ significantly increased survival and progression free survival. Preliminary data suggested benefit with both methylated and unmethylated O-6-methylguanine-DNA methyl-transferase (MGMT) promoter status. To date, however, there have been no studies to address the potential interactions of TTFields and TMZ. Thus, the effects of TTFields and TMZ were studied in vitro using patient-derived GBM stem-like cells (GSCs) including MGMT expressing (TMZ resistant: 12.1 and 22GSC) and non-MGMT expressing (TMZ sensitive: 33 and 114GSC) lines. Dose-response curves were constructed using cell proliferation and sphere-forming assays. Results demonstrated a ⩾10-fold increase in TMZ resistance of MGMT-expressing (12.1GSCs: IC50=160μM; 22GSCs: IC50=44μM) compared to MGMT non-expressing (33GSCs: IC50=1.5μM; 114GSCs: IC50=5.2μM) lines. TTFields inhibited 12.1 GSC proliferation at all tested doses (50–500kHz) with an optimal frequency of 200kHz. At 200kHz, TTFields inhibited proliferation and tumor sphere formation of both MGMT GSC subtypes at comparable levels (12.1GSC: 74±2.9% and 38±3.2%, respectively; 22GSC: 61±11% and 38±2.6%, respectively; 33GSC: 56±9.5% and 60±7.1%, respectively; 114 GSC: 79±3.5% and 41±4.3%, respectively). In combination, TTFields (200kHz) and TMZ showed an additive anti-neoplastic effect with equal efficacy for TTFields in both cell types (i.e., ± MGMT expression) with no effect on TMZ resistance. This is the first demonstration of the effects of TTFields on cancer stem cells. The expansion of such studies may have clinical implications.

Adenosine A3 receptor elicits chemoresistance mediated by multiple resistance-associated protein-1 in human glioblastoma stem-like cells.

MRP1 transporter correlates positively with glioma malignancy and the Multiple Drug Resistance (MDR) phenotype in Glioblastoma Multiforme (GBM). Evidence shows that the MRP1 transporter is controlled by the adenosine signalling axis. The aim of this study was to identify the role of adenosine on the MDR phenotype in Glioblastoma Stem-like Cells (GSCs), the cell population responsible for the tumorigenic and chemoresistance capabilities of this tumour. We found that GSCs have increased intrinsic capacity to generate extracellular adenosine, thus controlling MRP1 transporter expression and activity via activation of the adenosine A3 receptor (A3AR). We showed PI3K/Akt and MEK/ERK1/2 signaling pathways downstream A3AR to control MRP1 in GSCs. In vitro pharmacological blockade of A3AR had a chemosensitizing effect, enhancing the actions of antitumour drugs and decreasing cell viability and proliferation of GSCs. In addition, we produced an in vivo xenograft model by subcutaneous inoculation of human GSCs in NOD/SCID-IL2Rg null mice. Pharmacological blockade of A3AR generated a chemosensitizing effect, enhancing the effectiveness of the MRP1 transporter substrate, vincristine, reducing tumour size and the levels of CD44 and Nestin stem cell markers as well as the Ki-67 proliferation indicator. In conclusion, we demonstrated the chemosensitizing effect of A3AR blockade on GSCs.

Abstract 2845: PRMT5-PTEN molecular pathway regulates senescence and self-renewal of Glioblastoma stem-like cells

Abstract PURPOSE: Glioblastoma (GBM) represents the most common and aggressive histologic subtype among malignant astrocytoma and is associated with poor outcomes because of heterogeneous tumor cell population including mature non-stem like cell and immature stem-like cells within the tumor. Thus, it is critical to find new target-specific therapeutic modalities. Protein arginine methyltransferase enzyme 5 (PRMT5) regulates many cellular processes through its methylation activity and its overexpression in GBM is associated with more aggressive disease. Previously, we have shown that silencing of PRMT5 expression in differentiated GBM cell lines results in apoptosis and reduced tumor growth in mice. Here we report the critical role of PRMT5 in differentiated GBM non-stem-like cells (non-GSC) grown in serum and undifferentiated GBM stem-like cells (GSC) grown as neurospheres in vitro. METHODS: GSC grown in stem-cell media and non-GSC grown in serum were transfected with specified siRNAs. At appropriate time, these cells were tested for in vitro proliferation, self-renewal capacity, apoptosis, cell cycle progression, mRNA expression and protein expression. Also, using intracranial mouse xenograft, tumor forming ability of PRMT5-intacta and depleted GSC and non-GSC was determined. RESULTS: Our results uncover a very significant role for PRMT5 in GSC self-renewal capacity and proliferation. PRMT5 knockdown in non-GSC led to apoptosis, knockdown in GSC led to G1 cell cycle arrest through upregulation of p27 and hypophoshorylation of Rb protein, leading to senescence. Comparison of impact of PRMT5 on cellular signaling by Human Phospho-Kinase Array and ChIP-PCR revealed that unlike non-GSC, PRMT5 controls both AKT and ERK activity by direct repression of PTEN in GSC. In vivo transient depletion of PRMT5 decreased intracranial tumor size and growth rate in mice implanted with both primary tumor derived GSC and non-GSC. CONCLUSION: This is the first study to identify PTEN as a potential downstream target of PRMT5 and vital to support both mature and immature GBM tumor cell populations. Citation Format: Yeshavanth Kumar Banasavadi-Siddegowda, Luke Russell, Jianying Zhang, Vrajesh A. Karkhanis, Jaime Imitola, Robert Baiocchi, Balveen Kaur. PRMT5-PTEN molecular pathway regulates senescence and self-renewal of Glioblastoma stem-like cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2845.

Abstract 2523: Transcriptional profiling of glioblastoma stem-like cells reveals enrichment of ion channels with functional implications for malignancy

Abstract Glioblastoma Multiforme (GBM) is the most prevalent and aggressive form of cancer in the adult central nervous system. Ion channels are increasingly being linked to cancer progression through the regulation of cell proliferation and migration. However, the role of ion channels in GBM tumor formation and progression is not well understood. Glioblastoma stem-like cells (GSCs) are a source of tumor formation and recurrence, suggesting that ion channel expression may be perturbed in this population. Here, we used RNA-sequencing to assess the expression patterns of ion channels and transporters and identify uniquely enriched ion channels in GSCs. Twenty-two patient-derived GSC samples were expression profiled along with human neural stem cell (NSC) and astrocyte control cell populations. Differential expression analysis revealed a set of ion channels highly enriched in GSCs compared with controls. Real-time PCR analysis confirmed the increased expression of ion channels of interest across selected GSC lines compared to NSCs. The expression pattern of ion channel candidates was also more likely to be associated with distinct GBM molecular subtypes. Next, an integrative approach was taken to further identify ion channels unique to GSCs; the abovementioned findings were compared to results from transcriptome analyses of GBM bulk tumor cells (The Cancer Genome Atlas) and region-specific GBM cells (Ivy Glioblastoma Atlas Project). Ion channels that were identified in these analyses were associated with altered clinical outcomes. The functional implications of these expression changes were further assessed with targeted drug screening of GSCs and NSCs. Pharmacological antagonists of GSC-enriched ion channels suppressed stem cell viability. These antagonists similarly hindered BrdU incorporation of dividing GSCs, suggesting that these channels play a role in stem cell proliferative capacity. Finally, calcium imaging was used to test the real-time functional responses of GSCs to channel blockers, and these outcomes will be discussed. Collectively, these findings suggest the presence of ion channels that uniquely identify GSCs from other neural cell types and influence GSC proliferation, a hallmark of GBM tumor recurrence. Citation Format: Julia Pollak, Karan G. Rai, Patrick J. Paddison, Robert C. Rostomily, Jan-Marino Ramirez. Transcriptional profiling of glioblastoma stem-like cells reveals enrichment of ion channels with functional implications for malignancy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2523.