Glioblastoma Stem Research Articles

Holistic Analysis of Glioblastoma Stem Cell DNA Using Nanoengineered Plasmonic Metasensor for Glioblastoma Diagnosis.

The clinical relevance of liquid biopsy for glioblastoma (GBM) remains undetermined due to practical and biological limitations such as absence of a reliable GBM-specific biomarker, trace levels in circulation due to the blood-brain-barrier, and lack of a sensitive method to detect the trace levels of biomarkers. It is hypothesized that GBM stem cell (GSC)-associated cell free DNA can function as reliable biomarker for GBM because it accounts for tumor heterogeneity and provide accurate molecular information about the cancer. An integrative methodology is used for GBM diagnosis by utilizing the sub-single molecular sensitivity of nanoengineered plasmonic metasensors for real-time genomic profiling of GSC DNA. The nanoengineered metasensors can detect the rare circulating GSC-DNA accurately from just 5µL of blood and the test can be performed in under 10 min. Analysis of clinical serum samples from GBM patients and healthy volunteers demonstrates that the technology yielded an accurate classification of GBM in an independent validation cohort (accuracy 98.3%, specificity 100%). The methodology detects GBM-signatures from the patient blood rapidly within the half-life period of cfDNA in circulation, non-invasively and amplification-free with a high diagnostic accuracy. With clinical validation, this methodology can evolve as a clinically viable diagnostic tool for fatal and hard-to-detect cancer like GBM.

Antitumor Effects of Ral-GTPases Downregulation in Glioblastoma.

Glioblastoma (GBM) is the most common tumor in the central nervous system in adults. This neoplasia shows a high capacity of growth and spreading to the surrounding brain tissue, hindering its complete surgical resection. Therefore, the finding of new antitumor therapies for GBM treatment is a priority. We have previously described that cyclin D1-CDK4 promotes GBM dissemination through the activation of the small GTPases RalA and RalB. In this paper, we show that RalB GTPase is upregulated in primary GBM cells. We found that the downregulation of Ral GTPases, mainly RalB, prevents the proliferation of primary GBM cells and triggers a senescence-like response. Moreover, downregulation of RalA and RalB reduces the viability of GBM cells growing as tumorspheres, suggesting a possible role of these GTPases in the survival of GBM stem cells. By using mouse subcutaneous xenografts, we have corroborated the role of RalB in GBM growth in vivo. Finally, we have observed that the knockdown of RalB also inhibits cell growth in temozolomide-resistant GBM cells. Overall, our work shows that GBM cells are especially sensitive to Ral-GTPase availability. Therefore, we propose that the inactivation of Ral-GTPases may be a reliable therapeutic approach to prevent GBM progression and recurrence.

A rationally identified panel of microRNAs targets multiple oncogenic pathways to enhance chemotherapeutic effects in glioblastoma models

Glioblastoma (GBM) is the most common malignant brain tumor. Available treatments have limited success because most patients develop chemoresistance. Alternative strategies are required to improve anticancer effects of current chemotherapeutics while limiting resistance. Successful targeting of microRNAs (miRNAs) as regulators of gene expression can help reprogram GBM cells to better respond to chemotherapy. We aimed to identify a panel of miRNAs that target multiple oncogenic pathways to improve GBM therapy. We first identified differentially expressed miRNAs and tested if their target genes play central roles in GBM signaling pathways by analyzing data in the Gene Expression Omnibus and The Cancer Genome Atlas databases. We then studied the effects of different combinations of these miRNAs in GBM cells by delivering synthetic miRNAs using clinically compatible PLGA-PEG nanoparticles prior to treatment with temozolomide (TMZ) or doxorubicin (DOX). The successful miRNA panel was tested in mice bearing U87-MG cells co-treated with TMZ. We identified a panel of five miRNAs (miRNA-138, miRNA-139, miRNA-218, miRNA-490, and miRNA-21) and their oncogenic targets (CDK6, ZEB1, STAT3, TGIF2, and SMAD7) that cover four different signaling pathways (cell proliferation and apoptotic signaling, invasion and metastasis, cytokine signaling, and stemness) in GBM. We observed significant in vitro and in vivo enhancement of therapeutic efficiency of TMZ and DOX in GBM models. The proposed combination therapy using rationally selected miRNAs and chemotherapeutic drugs is effective owing to the ability of this specific miRNA panel to better target multiple genes associated with the hallmarks of cancer.

The Glycoprotein M6a Is Associated with Invasiveness and Radioresistance of Glioblastoma Stem Cells.

Systematic recurrence of glioblastoma (GB) despite surgery and chemo-radiotherapy is due to GB stem cells (GBSC), which are particularly invasive and radioresistant. Therefore, there is a need to identify new factors that might be targeted to decrease GBSC invasive capabilities as well as radioresistance. Patient-derived GBSC were used in this study to demonstrate a higher expression of the glycoprotein M6a (GPM6A) in invasive GBSC compared to non-invasive cells. In 3D invasion assays performed on primary neurospheres of GBSC, we showed that blocking GPM6A expression by siRNA significantly reduced cell invasion. We also demonstrated a high correlation of GPM6A with the oncogenic protein tyrosine phosphatase, PTPRZ1, which regulates GPM6A expression and cell invasion. The results of our study also show that GPM6A and PTPRZ1 are crucial for GBSC sphere formation. Finally, we demonstrated that targeting GPM6A or PTPRZ1 in GBSC increases the radiosensitivity of GBSC. Our results suggest that blocking GPM6A or PTPRZ1 could represent an interesting approach in the treatment of glioblastoma since it would simultaneously target proliferation, invasion, and radioresistance.

CD146 increases stemness and aggressiveness in glioblastoma and activates YAP signaling

Glioblastoma (GBM), a highly malignant and lethal brain tumor, is characterized by diffuse invasion into the brain and chemo-radiotherapy resistance resulting in poor prognosis. In this study, we examined the involvement of the cell adhesion molecule CD146/MCAM in regulating GBM aggressiveness. Analyses of GBM transcript expression databases revealed correlations of elevated CD146 levels with higher glioma grades, IDH-wildtype and unmethylated MGMT phenotypes, poor response to chemo-radiotherapy and worse overall survival. In a panel of GBM stem cells (GSCs) variable expression levels of CD146 were detected, which strongly increased upon adherent growth. CD146 was linked with mesenchymal transition since expression increased in TGF-ß-treated U-87MG cells. Ectopic overexpression of CD146/GFP in GG16 cells enhanced the mesenchymal phenotype and resulted in increased cell invasion. Conversely, GSC23-CD146 knockouts had decreased mesenchymal marker expression and reduced cell invasion in transwell and GBM-cortical assembloid assays. Moreover, using GSC23 xenografted zebrafish, we found that CD146 depletion resulted in more compact delineated tumor formation and reduced tumor cell dissemination. Stem cell marker expression and neurosphere formation assays showed that CD146 increased the stem cell potential of GSCs. Furthermore, CD146 mediated radioresistance by stimulating cell survival signaling through suppression of p53 expression and activation of NF-κB. Interestingly, CD146 was also identified as an inducer of the oncogenic Yes-associated protein (YAP). In conclusion, CD146 carries out various pro-tumorigenic roles in GBM involving its cell surface receptor function, which include the stimulation of mesenchymal and invasive properties, stemness, and radiotherapy resistance, thus providing an interesting target for therapy.

Abstract 5410: First, second, and third generation mTOR pathways inhibitors for treatment of glioblastoma

Abstract The Mechanistic Target of Rapamycin (mTOR), which functions in two distinct multiprotein complexes, mTORC1 and mTORC2, is a downstream signaling kinase in the PI3K/AKT pathway. mTOR is often deregulated in glioblastoma (GB), the most aggressive primary brain tumor in adults, due to the frequent loss of the tumor suppressor, PTEN. mTORC1 and mTORC2 control cell growth, proliferation, migration, survival, and stem cell regulation in response to nutrients and growth factors. Nutrient-sensitive mTORC1 regulates protein synthesis through the downstream substrate, 4EBP1 (also called EIF4EBP1), p70S6K1, and p70S6K2. On the other hand, growth factor-sensitive mTORC2 is associated with Rapamycin-insensitivity. The presence of stem cells within the tumor mass and the surrounding areas precludes the possibility of the elimination of GB. Furthermore, the substrate of activated AKT, the proline-rich AKT substrate of 40 kDa (PRAS40), is a surrogate marker of activated AKT in response to mTOR inhibition therapy in GB patients that regulates mTORC1. The aim of this study is to evaluate the efficacy of first (Rapamycin (RAPA)), second (Torin1, Torin2, and XL388), and third (Rapalink-1) mTOR inhibitors to suppress cell growth, dissemination, overcome drug resistance of GB cells, and alter self-renewal properties of GB stem cells. Results demonstrated: a significant number of tumors showed an increased expression of pAKTSer473 and pmTORSer2448, suggesting an overactivation of the mTOR pathway; co-expression of mTOR and stem cell marker, nestin, was evident in GB tumors, implying its role in stem cell regulation; Torin2, but not Torin1 or XL388, suppressed mTORC1 activity completely as shown by the reduced expression of its pS6KSer235/236 in a dose-dependent manner; Torin1, Torin2, and XL388 inhibited the phosphorylation of 4E-BP1 and PRAS40; Torin1, Torin2, and XL388 suppressed dephosphorylation of pAKTSer473; functional analysis revealed that RAPA, Torin1, Torin2, XL388, and Rapalink-1 effectively suppressed proliferation and S-phase entry; RAPA, Torin1, Torin2 and Rapalink-1 inhibited GB cell migration, while XL388 was ineffective; RAPA, Torin1, Torin2, and Rapalink-1 suppressed the self-renewal of GB stem cells and inhibited their proliferation, XL388 remained ineffective; drug resistance analysis revealed that Torin2 was most effective in eradicating GB tumor cells. These results suggested that RAPA, Torin1, Torin2, and Rapalink-1, but not XL388, are useful in suppressing the mTORC1 and mTORC2 activities, thereby inhibiting GB cell proliferation, dissemination, and inhibiting stem cell self-renewal. Therefore, the use of these mTOR inhibitors presents a promising treatment strategy for Glioblastoma. Citation Format: Jose F. Dominguez, Haylen Rosberger, Paul Garell, Chirag D. Gandhi, Meena Jhanwar-Uniyal. First, second, and third generation mTOR pathways inhibitors for treatment of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5410.

Abstract 3512: Immunotherapy by STING activation in a CRISPR induce mouse model of GBM decrease tumor progression by CD8 T-cells and altered vascularity

Abstract Intrinsic malignant brain tumors, such as glioblastoma (GBM) are frequently resistant to immunotherapy with few hyper-mutated glioblastomas showing response. Here we have applied a long-term treatment with STING agonist and PD-1 inhibitors alone and in combination towards eliciting enhanced host immune response to combat GBM progression. To address these questions we generated mouse models of GBM by intracranial implantation of a murine GBM stem cell line or orthotopic CRISPR induced mutation to astrocytes. Systemic administration of STING agonist crosses the blood-brain barrier and induces a type-1 IFN signature in the brain and this was further enhanced in the tumor milieu. Treatment outcomes were addressed by MRi and treated animals showed a significant reduction in tumor volume and hence improved survival in comparison to the animals in the control group. Histochemical evaluation of tumor samples from the animals that received treatment showed elevated necrosis and aberrant/leaky vasculature in comparison to the control tumor samples. The presence of cytotoxic T-cells were similar between the groups but activated PD-1 positive T-cells were increased in treated tumors. Ex-vivo analysis of isolated T-cells from tumor baring mice confirms cytotoxic T-cells targeting the GBM cells. In conclusion, application of STING agonist promotes alteration of vasculitis and priming of T-cells in GBM results in increased survival and decreased tumor progression. These results reveal that GBM can be targeted by immune therapy through systemic activation of STING/IFN pathway. Citation Format: Justin V. Joseph, Huiqiang Cai, Brian Hansen, Soren R. Paludan, Mikkel Vendelbo, Martin K. Thomsen. Immunotherapy by STING activation in a CRISPR induce mouse model of GBM decrease tumor progression by CD8 T-cells and altered vascularity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3512.

Abstract 3167: The scaffolding protein DLG5 is necessary to maintain Sonic Hedgehog signaling in glioblastoma stem cells and promote tumor growth

Abstract Proteins complexes that maintain the apico-basal polarity of epithelial cells and organize cell-signaling domains are necessary for normal tissue organization and therefore regarded as tumor suppressors. Accordingly, the scaffolding proteins that form the Scribble polarity complex (SCRIB, LLGL1, and DLG members) are usually downregulated in cancer, in particular in highly aggressive or invasive tumors. Because gliomas arise from non-epithelial precursors that are likely motile before transformation, the expression and functions of these scaffolding proteins in malignant brain tumors has remained almost entirely unexplored. Surprisingly, we have found that one member of the DLG family, DLG5, is highly upregulated in malignant gliomas compared to other solid tumors. Indeed, DLG5 was the predominant DLG member found in all molecular subtypes of glioblastoma (GBM). Knockdown of DLG5 reduced the viability, invasiveness, and self-renewal of GBM stem cells belonging to the proneural and mesenchymal molecular subtypes. As a result, intracranial tumors defficient in DLG5 were smaller and less proliferative than controls, resulting in extended animal survival. At the molecular level, DLG5 knockdown decreased the expression of stemness-related genes (SOX2, NANOG, POU5F1) but increased the expression of LLGL1, a component of the Scribble complex that is associated with neural precursor differentiation. These changes were accompanied by downregulation of Sonic Hedgehog (SHh) signaling, both in vitro and in vivo, which drives GBM cell stemness and proliferation. Moreover, DLG5-deficient GBM stem cells became insensitive to exogenous SHh and were unable to upregulate Gli1 or increase cell invasion in presence of added SHh, compared to control cells. We further confirmed that DLG5 downregulation increased the ubiquitination of Gli1-containing complexes as well as Gli1 proteasomal degradation, providing a suitable mechanism for the loss of SHh signaling and tumor stemness. These results describe for the first time the expression of a DLG family member in malignant gliomas and reveal a novel, polarity-independent, pro-tumoral function of DLG5, which differs from its proposed tumor-suppressor role in other solid tumors. Citation Format: Somanath Kundu, Mohan S. Nandhu, Sharon L. Longo, John A. Longo, Shawn Rai, Lawrence S. Chin, Timothy E. Richardson, Mariano S. Viapiano. The scaffolding protein DLG5 is necessary to maintain Sonic Hedgehog signaling in glioblastoma stem cells and promote tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3167.

Targeting Glioblastoma Stem Cells to Overcome Chemoresistance: An Overview of Current Therapeutic Strategies.

Glioblastoma (GBM) is the most malignant primary brain tumor. The current standard approach in GBM is surgery, followed by treatment with radiation and temozolomide (TMZ); however, GBM is highly resistant to current therapies, and the standard of care has not been revised over the last two decades, indicating an unmet need for new therapies. GBM stem cells (GSCs) are a major cause of chemoresistance due to their ability to confer heterogeneity and tumorigenic capacity. To improve patient outcomes and survival, it is necessary to understand the properties and mechanisms underlying GSC chemoresistance. In this review, we describe the current knowledge on various resistance mechanisms of GBM to therapeutic agents, with a special focus on TMZ, and summarize the recent findings on the intrinsic and extrinsic mechanisms of chemoresistance in GSCs. We also discuss novel therapeutic strategies, including molecular targeting, autophagy inhibition, oncolytic viral therapy, drug repositioning, and targeting of GSC niches, to eliminate GSCs, from basic research findings to ongoing clinical trials. Although the development of effective therapies for GBM is still challenging, this review provides a better understanding of GSCs and offers future directions for successful GBM therapy.

Functions of long non-coding RNA ROR in patient-derived glioblastoma cells

Glioblastoma (GBM) is the most frequent and aggressive primary brain cancer in adult patients. A variety of long non-coding RNAs play an important role in the pathogenesis of GBM, however the molecular functions of most of them still remain elusive. Here, we investigated linc-RoR (long intergenic non-protein coding RNA, regulator of reprogramming) using GBM neurospheres obtained from 12 different patients. We demonstrated that the highest level of this transcript is detected in cells with increased EGFR expression. According to our data, linc-RoR knockdown decreases cell proliferation, increases sensitivity to DNA damage, and downregulates the level of cancer stem cell (CSC) markers. On the other hand, linc-RoR overexpression promote cell growth and increases the proportion of CSCs. Analysis of RNA sequencing data revealed that linc-RoR affects expression of genes involved in the regulation of mitosis. In agreement with this observation, we have showen that the highest level of linc-RoR is detected in the G2/M phase of the cell cycle, when linc-RoR is localized on the chromosomes of dividing cells. Based on our results, we can propose that linc-RoR performs pro-oncogenic functions in human gliobalstoma cells, which may be associated with the regulation of mitotic progression and GBM stemness.

GSK3β palmitoylation mediated by ZDHHC4 promotes tumorigenicity of glioblastoma stem cells in temozolomide-resistant glioblastoma through the EZH2–STAT3 axis

Glioblastoma stem cells (GSCs) are a highly tumorigenic cell subgroup of glioblastoma (GBM). Glycogen synthase kinase 3β (GSK3β) is considered a key hub for promoting malignant phenotypes in GBM. However, the functional relationships between GSK3β and GSCs in GBM are unclear. Here, we found that GSK3β was noted as a substrate for ZDHHC4-mediated palmitoylation at the Cys14 residue, which enhanced GBM temozolomide (TMZ) resistance and GSC self-renewal. Clinically, the expression level of ZDHHC4 was upregulated in GBM, which significantly correlated with tumor grade and poor prognosis. The above phenotypes were based on decreasing p-Ser9 and increasing p-Tyr216 by GSK3β palmitoylation, which further activated the enhancer of the zeste homolog 2 (EZH2)–STAT3 pathway. Notably, STAT3 silencing also inhibited ZDHHC4 expression. This study revealed that GSK3β palmitoylation mediated by ZDHHC4 improved the stemness of TMZ-resistant GBM by activating the EZH2–STAT3 signaling axis, providing a new theoretical basis for further understanding the mechanism of TMZ resistance and recurrence after treatment.

Glioblastoma cell invasiveness and epithelial-to-mesenchymal transitioning are modulated by kinin receptors

Glioblastoma (GB) is the most aggressive primary tumor of central nervous system, where no efficient therapy has been found so far. This is due to high intra- and inter- tumor heterogeneity, fast invasion and the therapy-resistant subpopulation of GB stem cells and their plasticity. Tumor heterogeneity involves interactions among cancer cells and stromal cells, such as mesenchymal stem cells (MSCs), that are recruited into the tumor microenvironment and migrate to the tumor site. Here, we characterized the impact of kinin receptors on the interaction of MSCs with GB cells which is further enhanced by kinin receptor agonists. Interactions between GB cells and MSCs were studied in two- and three-dimensional co-cultures. Kinin receptor activity was modulated by selective agonists and antagonists to evaluate their influence on cell viability, cell-cell interactions, GB U87 cell invasiveness, and phenotype alterations. Tumor cell invasion was enhanced by the kinin-B2 receptor agonist bradykinin, while it was blocked by B2 receptor antagonists applied in U87 cells monoculture and in co-culture with MSCs. Kinin receptor inhibition correlated with enhanced direct U87/MSC interactions, such as heterotypic fusion, vesicle transfer and entosis. Furthermore, kinin receptor modulation influenced expression of F-actin expression and genes associated with epithelial-to-mesenchymal transition in U87 cells upon U87/MSCs co-cultures. Our data support ongoing investigations of kinin receptor inhibition as an adjuvant approach in GB therapy. MSC impacts on cancer progression need further investigation, as they may have a synergistic effect with kininergic receptor activation.

Fibroblast growth factor receptor 4 promotes glioblastoma progression: a central role of integrin-mediated cell invasiveness

Glioblastoma (GBM) is characterized by a particularly invasive phenotype, supported by oncogenic signals from the fibroblast growth factor (FGF)/ FGF receptor (FGFR) network. However, a possible role of FGFR4 remained elusive so far. Several transcriptomic glioma datasets were analyzed. An extended panel of primary surgical specimen-derived and immortalized GBM (stem)cell models and original tumor tissues were screened for FGFR4 expression. GBM models engineered for wild-type and dominant-negative FGFR4 overexpression were investigated regarding aggressiveness and xenograft formation. Gene set enrichment analyses of FGFR4-modulated GBM models were compared to patient-derived datasets. Despite widely absent in adult brain, FGFR4 mRNA was distinctly expressed in embryonic neural stem cells and significantly upregulated in glioblastoma. Pronounced FGFR4 overexpression defined a distinct GBM patient subgroup with dismal prognosis. Expression levels of FGFR4 and its specific ligands FGF19/FGF23 correlated both in vitro and in vivo and were progressively upregulated in the vast majority of recurrent tumors. Based on overexpression/blockade experiments in respective GBM models, a central pro-oncogenic function of FGFR4 concerning viability, adhesion, migration, and clonogenicity was identified. Expression of dominant-negative FGFR4 resulted in diminished (subcutaneous) or blocked (orthotopic) GBM xenograft formation in the mouse and reduced invasiveness in zebrafish xenotransplantation models. In vitro and in vivo data consistently revealed distinct FGFR4 and integrin/extracellular matrix interactions. Accordingly, FGFR4 blockade profoundly sensitized FGFR4-overexpressing GBM models towards integrin/focal adhesion kinase inhibitors. Collectively, FGFR4 overexpression contributes to the malignant phenotype of a highly aggressive GBM subgroup and is associated with integrin-related therapeutic vulnerabilities.

A Tumor-Homing Peptide Platform Enhances Drug Solubility, Improves Blood-Brain Barrier Permeability and Targets Glioblastoma.

Simple SummaryGlioblastoma (GBM) is a fatal and incurable brain cancer, and current treatment options have demonstrated limited success. Here, we describe the use of a dg-Bcan-Targeting-Peptide (BTP-7) that has BBB-penetrating properties and targets GBM. Conjugation of BTP-7 to an insoluble anti-cancer drug, camptothecin (CPT), improves drug solubility in aqueous solution, retains drug efficacy against patient-derived GBM stem cells (GSC), enhances BBB permeability, and enables therapeutic targeting to intracranial patient-derived GBM xenograft in mice, leading to higher toxicity in GBM cells compared to normal brain tissues and prolonged animal survival. This work demonstrates a proof-of-concept for BTP-7 as a tumor-targeting peptide for therapeutic delivery to GBM. Background: Glioblastoma (GBM) is the most common and deadliest malignant primary brain tumor, contributing significant morbidity and mortality among patients. As current standard-of-care demonstrates limited success, the development of new efficacious GBM therapeutics is urgently needed. Major challenges in advancing GBM chemotherapy include poor bioavailability, lack of tumor selectivity leading to undesired side effects, poor permeability across the blood–brain barrier (BBB), and extensive intratumoral heterogeneity. Methods: We have previously identified a small, soluble peptide (BTP-7) that is able to cross the BBB and target the human GBM extracellular matrix (ECM). Here, we covalently attached BTP-7 to an insoluble anti-cancer drug, camptothecin (CPT). Results: We demonstrate that conjugation of BTP-7 to CPT improves drug solubility in aqueous solution, retains drug efficacy against patient-derived GBM stem cells (GSC), enhances BBB permeability, and enables therapeutic targeting to intracranial GBM, leading to higher toxicity in GBM cells compared to normal brain tissues, and ultimately prolongs survival in mice bearing intracranial patient-derived GBM xenograft. Conclusion: BTP-7 is a new modality that opens the door to possibilities for GBM-targeted therapeutic approaches.

Crosstalk between β-Catenin and CCL2 Drives Migration of Monocytes towards Glioblastoma Cells.

Isocitrate dehydrogenase (IDH)-wildtype glioblastoma (GBM) is a fast growing and highly heterogeneous tumor, often characterized by the presence of glioblastoma stem cells (GSCs). The plasticity of GSCs results in therapy resistance and impairs anti-tumor immune response by influencing immune cells in the tumor microenvironment (TME). Previously, β-catenin was associated with stemness in GBM as well as with immune escape mechanisms. Here, we investigated the effect of β-catenin on attracting monocytes towards GBM cells. In addition, we evaluated whether CCL2 is involved in β-catenin crosstalk between monocytes and tumor cells. Our analysis revealed that shRNA targeting β-catenin in GBMs reduces monocytes attraction and impacts CCL2 secretion. The addition of recombinant CCL2 restores peripheral blood mononuclear cells (PBMC) migration towards medium (TCM) conditioned by shβ-catenin GBM cells. CCL2 knockdown in GBM cells shows similar effects and reduces monocyte migration to a similar extent as β-catenin knockdown. When investigating the effect of CCL2 on β-catenin activity, we found that CCL2 modulates components of the Wnt/β-catenin pathway and alters the clonogenicity of GBM cells. In addition, the pharmacological β-catenin inhibitor MSAB reduces active β-catenin, downregulates the expression of associated genes and alters CCL2 secretion. Taken together, we showed that β-catenin plays an important role in attracting monocytes towards GBM cells in vitro. We hypothesize that the interactions between β-catenin and CCL2 contribute to maintenance of GSCs via modulating immune cell interaction and promoting GBM growth and recurrence.

FTO gene polymorphisms and hepatoblastoma susceptibility among Chinese children

ABSTRACT Current knowledge on the etiology of hepatoblastoma remains limited. FTO gene has been documented as a susceptibility gene for several types of cancer. However, its role has not been characterized in hepatoblastoma. Herein, we intended to explore whether FTO gene single nucleotide polymorphisms (SNPs) contribute to the risk of hepatoblastoma. A multi-center case–control study was conducted including 358 cases and 1512 controls recruited from the night hospitals in China. Odds ratios (ORs) and 95% confidence intervals (CIs), for the association of FTO gene SNPs with hepatoblastoma risk, were estimated using conditional logistic regression models, adjusted for relevant confounding variables. Four SNPs (rs1477196 G > A, rs9939609 T > A, rs7206790 C > G, and rs8047395 A > G) in the FTO gene were genotyped. We detected a significant association between rs9939609 T > A and decreased risk of hepatoblastoma (TA vs. TT: adjust OR = 0.73, 95% CI = 0.54–0.99, P = 0.041; TA/AA vs. TT: adjust OR = 0.73, 95% CI = 0.55–0.97, P = 0.032). Compared to 0–3 protective genotypes, carriers with four protective genotypes showed enough strength to protect from hepatoblastoma (adjust OR = 0.65, 95% CI = 0.47–0.91, P = 0.012). In stratification analysis, we also detected a significantly decreased risk of hepatoblastoma in subjects with rs9939609 TA/AA or with four protective genotypes in some subgroups. Our results provided some clues for an association of FTO gene SNPs with hepatoblastoma risk in Chinese children. Abbreviations: GWAS, genome-wide association study; FTO, The fat mass and obesity-associated gene; SNP, single nucleotide polymorphism; m6A, N6-methyladenosine; mRNA, messenger RNA; LD, linkage disequilibrium; HWE, Hardy-Weinberg equilibrium; OR, odds ratio; CI, confidence interval; AML, acute myeloid leukemia; GSC, glioblastoma stem(-like) cell; HER2, human epidermal growth factor receptor 2.

Purine Synthesis Inhibitor L-Alanosine Impairs Mitochondrial Function and Stemness of Brain Tumor Initiating Cells.

Glioblastoma (GBM) is a lethal brain cancer exhibiting high levels of drug resistance, a feature partially imparted by tumor cell stemness. Recent work shows that homozygous MTAP deletion, a genetic alteration occurring in about half of all GBMs, promotes stemness in GBM cells. Exploiting MTAP loss-conferred deficiency in purine salvage, we demonstrate that purine blockade via treatment with L-Alanosine (ALA), an inhibitor of de novo purine synthesis, attenuates stemness of MTAP-deficient GBM cells. This ALA-induced reduction in stemness is mediated in part by compromised mitochondrial function, highlighted by ALA-induced elimination of mitochondrial spare respiratory capacity. Notably, these effects of ALA are apparent even when the treatment was transient and with a low dose. Finally, in agreement with diminished stemness and compromised mitochondrial function, we show that ALA sensitizes GBM cells to temozolomide (TMZ) in vitro and in an orthotopic GBM model. Collectively, these results identify purine supply as an essential component in maintaining mitochondrial function in GBM cells and highlight a critical role of mitochondrial function in sustaining GBM stemness. We propose that purine synthesis inhibition can be beneficial in combination with the standard of care for MTAP-deficient GBMs, and that it may be feasible to achieve this benefit without inflicting major toxicity.

Impact of Regorafenib on Endothelial Transdifferentiation of Glioblastoma Stem-like Cells.

Simple SummaryGlioblastomas (GBM) are the most frequent and aggressive adult brain tumours with a poor prognosis despite heavy therapy that combines surgical resection and radio-chemotherapy. There is a real therapeutic failure concerning these tumours and it highlights the need for new efficient and usable molecules in these particular and not easily reachable cancers. GBM are also characterised by abnormal and abundant vascularisation, of which some vessels originate from transdifferentiation of GBM stem cells (GSC) to tumour-derived endothelial cells (TDEC). As this mechanism was described as essential for tumour growth, we evaluated the impact of regorafenib, a multikinase inhibitor with anti-angiogenic and anti-tumourigenic activity on transdifferentiation but also directly on GSC. Regorafenib significantly decreases GSC proliferation in vitro but also in vivo. Regorafenib also inhibits transdifferentiation by decreasing phenotypic and functional endothelial features of TDEC obtained from non-irradiated GSC but also of TDEC obtained from irradiated GSC. All these results confirm that regorafenib clearly impacts GSC tumour formation and transdifferentiation and may therefore be a promising therapeutic option in combination with chemo/radiotherapy for the treatment of these highly aggressive brain tumours.Glioblastomas (GBM) are aggressive brain tumours with a poor prognosis despite heavy therapy that combines surgical resection and radio-chemotherapy. The presence of a subpopulation of GBM stem cells (GSC) contributes to tumour aggressiveness, resistance and recurrence. Moreover, GBM are characterised by abnormal, abundant vascularisation. Previous studies have shown that GSC are directly involved in new vessel formation via their transdifferentiation into tumour-derived endothelial cells (TDEC) and that irradiation (IR) potentiates the pro-angiogenic capacity of TDEC via the Tie2 signalling pathway. We therefore investigated the impact of regorafenib, a multikinase inhibitor with anti-angiogenic and anti-tumourigenic activity, on GSC and TDEC obtained from irradiated GSC (TDEC IR+) or non-irradiated GSC (TDEC). Regorafenib significantly decreases GSC neurosphere formation in vitro and inhibits tumour formation in the orthotopic xenograft model. Regorafenib also inhibits transdifferentiation by decreasing CD31 expression, CD31+ cell count, pseudotube formation in vitro and the formation of functional blood vessels in vivo of TDEC and TDEC IR+. All of these results confirm that regorafenib clearly impacts GSC tumour formation and transdifferentiation and may therefore be a promising therapeutic option in combination with chemo/radiotherapy for the treatment of highly aggressive brain tumours.

ADAR1-mediated RNA editing links ganglioside catabolism to glioblastoma stem cell maintenance.

Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor, containing GBM stem cells (GSCs) that contribute to therapeutic resistance and relapse. Exposing potential GSC vulnerabilities may provide therapeutic strategies against GBM. Here, we interrogated the role of adenosine-to-inosine (A-to-I) RNA editing mediated by adenosine deaminase acting on RNA 1 (ADAR1) in GSCs and found that both ADAR1 and global RNA editomes were elevated in GSCs compared with normal neural stem cells. ADAR1 inactivation or blocking of the upstream JAK/STAT pathway through TYK2 inhibition impaired GSC self-renewal and stemness. Downstream of ADAR1, RNA editing of the 3′-UTR of GM2A, a key ganglioside catabolism activator, proved to be critical, as interference with ganglioside catabolism and disruption of ADAR1 showed a similar functional impact on GSCs. These findings reveal that RNA editing links ganglioside catabolism to GSC self-renewal and stemness, exposing a potential vulnerability of GBM for therapeutic intervention.

The endosomal pH regulator NHE9 is a driver of stemness in glioblastoma.

A small population of self-renewing stem cells initiate tumors and maintain therapeutic resistance in glioblastoma (GBM). Given the limited treatment options and dismal prognosis for this disease, there is urgent need to identify drivers of stem cells that could be druggable targets. Previous work showed that the endosomal pH regulator NHE9 is upregulated in GBM and correlates with worse survival prognosis. Here, we probed for aberrant signaling pathways in patient-derived GBM cells and found that NHE9 increases cell surface expression and phosphorylation of multiple receptor tyrosine kinases (RTKs) by promoting their escape from lysosomal degradation. Downstream of NHE9-mediated receptor activation, oncogenic signaling pathways converged on the JAK2-STAT3 transduction axis to induce pluripotency genes Oct4 and Nanog and suppress markers of glial differentiation. We used both genetic and chemical approaches to query the role of endosomal pH in GBM phenotypes. Loss-of-function mutations in NHE9 that failed to alkalinize endosomal lumen did not increase self-renewal capacity of gliomaspheres in vitro. However, monensin, a chemical mimetic of Na+/H+ exchanger activity, and the H+ pump inhibitor bafilomycin bypassed NHE9 to directly alkalinize the endosomal lumen resulting in stabilization of RTKs and induction of Oct4 and Nanog. Using orthotopic models of primary GBM cells we found that NHE9 increased tumor initiation in vivo. We propose that NHE9 initiates inside-out signaling from the endosomal lumen, distinct from the established effects of cytosolic and extracellular pH on tumorigenesis. Endosomal pH may be an attractive therapeutic target that diminishes stemness in GBM, agnostic of specific receptor subtype.