The Impact of A3AR Antagonism on the Differential Expression of Chemoresistance-Related Genes in Glioblastoma Stem-like Cells.

Glioblastoma (GB) is the most aggressive and common primary malignant tumor of the brain and central nervous system. Without treatment, the average patient survival time is about six months, which can be extended to fifteen months with multimodal therapies. The chemoresistance observed in GB is, in part, attributed to the presence of a subpopulation of glioblastoma-like stem cells (GSCs) that are characterized by heightened tumorigenic capacity and chemoresistance. GSCs are situated in hypoxic tumor niches, where they sustain and promote the stem-like phenotype and have also been correlated with high chemoresistance. GSCs have the particularity of generating high levels of extracellular adenosine (ADO), which causes the activation of the A3 adenosine receptor (A3AR) with a consequent increase in the expression and activity of genes related to chemoresistance. Therefore, targeting its components is a promising alternative for treating GB. This analysis determined genes that were up- and downregulated due to A3AR blockades under both normoxic and hypoxic conditions. In addition, possible candidates associated with chemoresistance that were positively regulated by hypoxia and negatively regulated by A3AR blockades in the same condition were analyzed. We detected three potential candidate genes that were regulated by the A3AR antagonist MRS1220 under hypoxic conditions: LIMD1, TRIB2, and TGFB1. Finally, the selected markers were correlated with hypoxia-inducible genes and with the expression of adenosine-producing ectonucleotidases. In conclusion, we detected that hypoxic conditions generate extensive differential gene expression in GSCs, increasing the expression of genes associated with chemoresistance. Furthermore, we observed that MRS1220 could regulate the expression of LIMD1, TRIB2, and TGFB1, which are involved in chemoresistance and correlate with a poor prognosis, hypoxia, and purinergic signaling.

Proteomic, Metabolomic, and Fatty Acid Profiling of Small Extracellular Vesicles from Glioblastoma Stem-Like Cells and Their Role in Tumor Heterogeneity.

Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-like cells (GSCs) contributes to the heterogeneous nature of the disease and makes developing effective therapies challenging. Glioblastoma cells have been shown to influence their environment by releasing biological nanostructures known as extracellular vesicles (EVs). Here, we investigated the role of GSC-derived nanosized EVs (<200 nm) in glioblastoma heterogeneity, plasticity, and aggressiveness, with a particular focus on their protein, metabolite, and fatty acid content. We showed that conditioned medium and small extracellular vesicles (sEVs) derived from cells of one glioblastoma subtype induced transcriptomic and proteomic changes in cells of another subtype. We found that GSC-derived sEVs are enriched in proteins playing a role in the transmembrane transport of amino acids, carboxylic acids, and organic acids, growth factor binding, and metabolites associated with amino acid, carboxylic acid, and sugar metabolism. This suggests a dual role of GSC-derived sEVs in supplying neighboring GSCs with valuable metabolites and proteins responsible for their transport. Moreover, GSC-derived sEVs were enriched in saturated fatty acids, while their respective cells were high in unsaturated fatty acids, supporting that the loading of biological cargos into sEVs is a highly regulated process and that GSC-derived sEVs could be sources of saturated fatty acids for the maintenance of glioblastoma cell metabolism. Interestingly, sEVs isolated from GSCs of the proneural and mesenchymal subtypes are enriched in specific sets of proteins, metabolites, and fatty acids, suggesting a molecular collaboration between transcriptionally different glioblastoma cells. In summary, this study revealed the complexity of GSC-derived sEVs and unveiled their potential contribution to tumor heterogeneity and critical cellular processes commonly deregulated in glioblastoma.

Nuclear Glycoprotein A Repetitions Predominant (GARP) Is a Common Trait of Glioblastoma Stem-like Cells and Correlates with Poor Survival in Glioblastoma Patients.

Glioblastoma (GB) is notoriously resistant to therapy. GB genesis and progression are driven by glioblastoma stem-like cells (GSCs). One goal for improving treatment efficacy and patient outcomes is targeting GSCs. Currently, there are no universal markers for GSCs. Glycoprotein A repetitions predominant (GARP), an anti-inflammatory protein expressed by activated regulatory T cells, was identified as a possible marker for GSCs. This study evaluated GARP for the detection of human GSCs utilizing a multidimensional experimental design that replicated several features of GB: (1) intratumoral heterogeneity, (2) cellular hierarchy (GSCs with varied degrees of self-renewal and differentiation), and (3) longitudinal GSC evolution during GB recurrence (GSCs from patient-matched newly diagnosed and recurrent GB). Our results indicate that GARP is expressed by GSCs across various cellular states and disease stages. GSCs with an increased GARP expression had reduced self-renewal but no alterations in proliferative capacity or differentiation commitment. Rather, GARP correlated inversely with the expression of GFAP and PDGFR-α, markers of astrocyte or oligodendrocyte differentiation. GARP had an abnormal nuclear localization (GARPNU+) in GSCs and was negatively associated with patient survival. The uniformity of GARP/GARPNU+ expression across different types of GSCs suggests a potential use of GARP as a marker to identify GSCs.

Development of a Synthetic, Injectable Hydrogel to Capture Residual Glioblastoma and Glioblastoma Stem-Like Cells with CXCL12-Mediated Chemotaxis.

Glioblastoma (GBM), characterized by high infiltrative capacity, is the most common and deadly type of primary brain tumor in adults. GBM cells, including therapy-resistant glioblastoma stem-like cells (GSCs), invade the healthy brain parenchyma to form secondary tumors even after patients undergo surgical resection and chemoradiotherapy. New techniques are therefore urgently needed to eradicate these residual tumor cells. A thiol-Michael addition injectable hydrogel for compatibility with GBM therapy is previously characterized and optimized. This study aims to develop the hydrogel further to capture GBM/GSCs through CXCL12-mediated chemotaxis. The release kinetics of hydrogel payloads are investigated, migration and invasion assays in response to chemoattractants are performed, and the GBM-hydrogel interactions in vitro are studied. With a novel dual-layer hydrogel platform, it is demonstrated that CXCL12 released from the synthetic hydrogel can induce the migration of U251 GBM cells and GSCs from the extracellular matrix microenvironment and promote invasion into the synthetic hydrogel via amoeboid migration. The survival of GBM cells entrapped deep into the synthetic hydrogel is limited, while live cells near the surface reinforce the hydrogel through fibronectin deposition. This synthetic hydrogel, therefore, demonstrates a promising method to attract and capture migratory GBM cells and GSCs responsive to CXCL12 chemotaxis.

Glioblastoma Microenvironment and Invasiveness: New Insights and Therapeutic Targets.

Glioblastoma (GBM) is the most common and malignant primary brain cancer in adults. Without treatment the mean patient survival is approximately 6 months, which can be extended to 15 months with the use of multimodal therapies. The low effectiveness of GBM therapies is mainly due to the tumor infiltration into the healthy brain tissue, which depends on GBM cells' interaction with the tumor microenvironment (TME). The interaction of GBM cells with the TME involves cellular components such as stem-like cells, glia, endothelial cells, and non-cellular components such as the extracellular matrix, enhanced hypoxia, and soluble factors such as adenosine, which promote GBM's invasiveness. However, here we highlight the role of 3D patient-derived glioblastoma organoids cultures as a new platform for study of the modeling of TME and invasiveness. In this review, the mechanisms involved in GBM-microenvironment interaction are described and discussed, proposing potential prognosis biomarkers and new therapeutic targets.

Identification of Dysregulated microRNAs in Glioblastoma Stem-like Cells.

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults. Despite multimodal therapy, median survival is poor at 12-15 months. At the molecular level, radio-/chemoresistance and resulting tumor progression are attributed to a small fraction of tumor cells, termed glioblastoma stem-like cells (GSCs). These CD133-expressing, self-renewing cells display the properties of multi-lineage differentiation, resulting in the heterogenous composition of GBM. MicroRNAs (miRNAs) as regulators of gene expression at the post-transcriptional level can alter many pathways pivotal to cancer stem cell fate. This study explored changes in the miRNA expression profiles in patient-derived GSCs altered on differentiation into glial fiber acid protein (GFAP)-expressing, astrocytic tumor cells using a polymerase chain reaction (PCR) array. Initially, 22 miRNAs showed higher expression in GSCs and 9 miRNAs in differentiated cells. The two most downregulated miRNAs in differentiated GSCs were miR-17-5p and miR-425-5p, whilst the most upregulated miRNAs were miR-223-3p and let-7-5p. Among those, miR-425-5p showed the highest consistency in an upregulation in all three GSCs. By transfection of a 425-5p miRNA mimic, we demonstrated downregulation of the GFAP protein in differentiated patient-derived GBM cells, providing potential evidence for direct regulation of miRNAs in the GSC/GBM cell transition.

Ror1 is expressed inducibly by Notch and hypoxia signaling and regulates stem cell-like property of glioblastoma cells.

Ror1 plays a crucial role in cancer progression by regulating cell proliferation and migration. Ror1 is expressed abundantly in various types of cancer cells and cancer stem-like cells. However, the molecular mechanisms regulating expression of Ror1 in these cells remain largely unknown. Ror1 and its putative ligand Wnt5a are expressed highly in malignant gliomas, especially in glioblastomas, and the extents of Ror1 expression are correlated positively with poorer prognosis in patients with gliomas. We show that Ror1 expression can be upregulated in glioblastoma cells under spheroid culture, but not adherent culture conditions. Notch and hypoxia signaling pathways have been shown to be activated in spheroid-forming glioblastoma stem-like cells (GSCs), and Ror1 expression in glioblastoma cells is indeed suppressed by inhibiting either Notch or hypoxia signaling. Meanwhile, either forced expression of the Notch intracellular domain (NICD) in or hypoxic culture of glioblastoma cells result in enhanced expression of Ror1 in the cells. Consistently, we show that both NICD and hypoxia-inducible factor 1 alpha bind to upstream regions within the Ror1 gene more efficiently in GSCs under spheroid culture conditions. Furthermore, we provide evidence indicating that binding of Wnt5a to Ror1, upregulated by Notch and hypoxia signaling pathways in GSCs, might promote their spheroid-forming ability. Collectively, these findings indicate for the first time that Notch and hypoxia signaling pathways can elicit a Wnt5a-Ror1 axis through transcriptional activation of Ror1 in glioblastoma cells, thereby promoting their stem cell-like property.

Synergistic Anticancer Effect of a Combination of Berbamine and Arcyriaflavin A against Glioblastoma Stem-like Cells.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor. Relapse is frequent and rapid due to glioblastoma stem-like cells (GSCs) that induce tumor initiation, drug resistance, high cancer invasion, immune evasion, and recurrence. Therefore, suppression of GSCs is a powerful therapeutic approach for GBM treatment. Natural compounds berbamine and arcyriaflavin A (ArcA) are known to possess anticancer activity by targeting calcium/calmodulin-dependent protein kinase II gamma (CaMKIIγ) and cyclin-dependent kinase 4 (CDK4), respectively. In this study, we evaluated the effects of concurrent treatment with both compounds on GSCs. Combined treatment with berbamine and ArcA synergistically inhibited cell viability and tumorsphere formation in U87MG- and C6-drived GSCs. Furthermore, simultaneous administration of both compounds potently inhibited tumor growth in a U87MG GSC-grafted chick embryo chorioallantoic membrane (CAM) model. Notably, the synergistic anticancer effect of berbamine and ArcA on GSC growth is associated with the promotion of reactive oxygen species (ROS)- and calcium-dependent apoptosis via strong activation of the p53-mediated caspase cascade. Moreover, co-treatment with both compounds significantly reduced the expression levels of key GSC markers, including CD133, integrin α6, aldehyde dehydrogenase 1A1 (ALDH1A1), Nanog, Sox2, and Oct4. The combined effect of berbamine and ArcA on GSC growth also resulted in downregulation of cell cycle regulatory proteins, such as cyclins and CDKs, by potent inactivation of the CaMKIIγ-mediated STAT3/AKT/ERK1/2 signaling pathway. In addition, a genetic knockdown study using small interfering RNAs (siRNAs) targeting either CaMKIIγ or CDK4 demonstrated that the synergistic anticancer effect of the two compounds on GSCs resulted from dual inhibition of CaMKIIγ and CDK4. Collectively, our findings suggest that a novel combination therapy involving berbamine and ArcA could effectively eradicate GSCs.

Glioblastoma-Specific Strategies of Vascularization: Implications in Anti-Angiogenic Therapy Resistance.

Angiogenesis has long been implicated as a crucial process in GBM growth and progression. GBM can adopt several strategies to build up its abundant and aberrant vasculature. Targeting GBM angiogenesis has gained more and more attention in anti-cancer therapy, and many strategies have been developed to interfere with this hallmark. However, recent findings reveal that the effects of anti-angiogenic treatments are temporally limited and that tumors become refractory to therapy and more aggressive. In this review, we summarize the GBM-associated neovascularization processes and their implication in drug resistance mechanisms underlying the transient efficacy of current anti-angiogenic therapies. Moreover, we describe potential strategies and perspectives to overcome the mechanisms adopted by GBM to develop resistance to anti-angiogenic therapy as new potential therapeutic approaches.

The low affinity A2B adenosine receptor enhances migratory and invasive capacity in vitro and angiogenesis in vivo of glioblastoma stem-like cells.

Glioblastoma (GBM) is the most common and deadly malignant brain tumor, with a median survival of 15 to 17 months for a patient. GBM contains a cellular subpopulation known as GBM stem-like cells (GSCs) that persist in hypoxic niches and are capable of infiltrating into healthy brain tissue. For this reason, GSCs are considered one of the main culprits for GBM recurrence. A hypoxic microenvironment increases extracellular adenosine levels, activating the low affinity A2B adenosine receptor (A2BAR). Adenosine, through A2BAR, is capable of modulating invasiveness. However, its role in the invasion/migration of hypoxic-GSCs is still unknown. This study aims to understand the importance of A2BAR in modulating the migratory/invasive capacity of GSCs under hypoxia. Data analysis from The Cancer Genome Atlas (TCGA) program correlates A2BAR expression with high-grade glioma and hypoxic necrotic areas. U87MG and primary culture-derived GSCs under hypoxic conditions (0.5% O2) increased A2BAR mRNA and protein levels. As expected, the migratory and invasive capacity of GSCs increased under hypoxia, which was counteracted by blocking A2BAR, through the downregulation of MMP9 activity and epithelial-mesenchymal transition marker expression. Finally, in a xenograft mouse model, we demonstrate that treatment with MRS1754 did not affect the tumor volume but could decrease blood vessel formation and VEGF expression. Our results suggest that extracellular adenosine, through the activation of A2BAR, enhances the migratory and invasive capacity of GSCs in vitro under hypoxic conditions. Targeting A2BAR can be an effective therapy for GBM recurrence.

Nanobody-based retargeting of an oncolytic herpesvirus for eliminating CXCR4+ GBM cells: A proof of principle.

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults, which remains difficult to cure. The very high recurrence rate has been partly attributed to the presence of GBM stem-like cells (GSCs) within the tumors, which have been associated with elevated chemokine receptor 4 (CXCR4) expression. CXCR4 is frequently overexpressed in cancer tissues, including GBM, and usually correlates with a poor prognosis. We have created a CXCR4-retargeted oncolytic herpesvirus (oHSV) by insertion of an anti-human CXCR4 nanobody in glycoprotein D of an attenuated HSV-1 (ΔICP34.5, ΔICP6, and ΔICP47), thereby describing a proof of principle for the use of nanobodies to target oHSVs toward specific cellular entities. Moreover, this virus has been armed with a transgene expressing a soluble form of TRAIL to trigger apoptosis. In vitro, this oHSV infects U87MG CXCR4+ and patient-derived GSCs in a CXCR4-dependent manner and, when armed, triggers apoptosis. In a U87MG CXCR4+ orthotopic xenograft mouse model, this oHSV slows down tumor growth and significantly improves mice survival. Customizing oHSVs with diverse nanobodies for targeting multiple proteins appears as an interesting approach for tackling the heterogeneity of GBM, especially GSCs. Altogether, our study must be considered as a proof of principle and a first step toward personalized GBM virotherapies to complement current treatments.

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

[This corrects the article DOI: 10.3389/fonc.2020.608082.].

A Tumor Suppressor Gene, N-myc Downstream-Regulated Gene 1 (NDRG1), in Gliomas and Glioblastomas.

The development of potent and selective therapeutic approaches to glioblastoma (GBM) requires the identification of molecular pathways that critically regulate the survival and proliferation of GBM. Glioblastoma stem-like cells (GSCs) possess stem-cell-like properties, self-renewal, and differentiation into multiple neural cell lineages. From a clinical point of view, GSCs have been reported to resist radiation and chemotherapy. GSCs are influenced by the microenvironment, especially the hypoxic condition. N-myc downstream-regulated gene 1 (NDRG1) is a tumor suppressor with the potential to suppress the proliferation, invasion, and migration of cancer cells. Previous studies have reported that deregulated expression of NDRG1 affects tumor growth and clinical outcomes of patients with GBM. This literature review aimed to clarify the critical role of NDRG1 in tumorigenesis and acquirement of resistance for anti-GBM therapies, further to discussing the possibility and efficacy of NDRG1 as a novel target of treatment for GBM. The present review was conducted by searching the PubMed and Scopus databases. The search was conducted in February 2022. We review current knowledge on the regulation and signaling of NDRG1 in neuro-oncology. Finally, the role of NDRG1 in GBM and potential clinical applications are discussed.

Discovery of a New CaMKII-Targeted Synthetic Lethal Therapy against Glioblastoma Stem-like Cells.

Glioblastoma stem-like cells (GSCs) drive tumor initiation, cancer invasion, immune evasion, and therapeutic resistance and are thus a key therapeutic target for improving treatment for glioblastoma multiforme (GBM). We previously identified calcium/calmodulin-dependent protein kinase II (CaMKII) as an emerging molecular target for eliminating GSCs. In this study, we aim to explore a new CaMKII-targeted synthetic lethal therapy for GSCs. Through high-throughput drug combination screening using CaMKII inhibitors and a bioactive compound library in GSCs, neurokinin 1 receptor (NK1R) inhibitors such as SR 140333 and aprepitant are found to be potential anticancer agents that exhibit chemical synthetic lethal interactions with CaMKII inhibitors, including hydrazinobenzoylcurcumin (HBC), berbamine, and KN93. Combined treatment with NK1R and CaMKII inhibitors markedly suppresses the viability and neurosphere formation of U87MG- and U373MG-derived GSCs. In addition, the combination of HBC and NK1R inhibitors significantly inhibits U87MG GSC tumor growth in a chick embryo chorioallantoic membrane (CAM) model. Furthermore, the synthetic lethal interaction is validated using RNA interference of CaMKIIγ and NK1R. Notably, the synthetic lethal effects in GSCs are associated with the activation of caspase-mediated apoptosis by inducing p53 expression and reactive oxygen species generation, as well as the suppression of stemness marker expression by reducing nuclear factor-kappa B (NF-κB) activity. This follows the downregulation of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling and a decrease in intracellular calcium concentration. Moreover, NK1R affects CaMKIIγ activation. These findings demonstrate that NK1R is a potential synthetic lethal partner of CaMKII that is involved in eradicating GSCs, and they suggest a new CaMKII-targeted combination therapy for treating GBM.

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

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.

Adapt to Persist: Glioblastoma Microenvironment and Epigenetic Regulation on Cell Plasticity.

Glioblastoma (GBM) is the most frequent and aggressive brain tumor, characterized by great resistance to treatments, as well as inter- and intra-tumoral heterogeneity. GBM exhibits infiltration, vascularization and hypoxia-associated necrosis, characteristics that shape a unique microenvironment in which diverse cell types are integrated. A subpopulation of cells denominated GBM stem-like cells (GSCs) exhibits multipotency and self-renewal capacity. GSCs are considered the conductors of tumor progression due to their high tumorigenic capacity, enhanced proliferation, invasion and therapeutic resistance compared to non-GSCs cells. GSCs have been classified into two molecular subtypes: proneural and mesenchymal, the latter showing a more aggressive phenotype. Tumor microenvironment and therapy can induce a proneural-to-mesenchymal transition, as a mechanism of adaptation and resistance to treatments. In addition, GSCs can transition between quiescent and proliferative substates, allowing them to persist in different niches and adapt to different stages of tumor progression. Three niches have been described for GSCs: hypoxic/necrotic, invasive and perivascular, enhancing metabolic changes and cellular interactions shaping GSCs phenotype through metabolic changes and cellular interactions that favor their stemness. The phenotypic flexibility of GSCs to adapt to each niche is modulated by dynamic epigenetic modifications. Methylases, demethylases and histone deacetylase are deregulated in GSCs, allowing them to unlock transcriptional programs that are necessary for cell survival and plasticity. In this review, we described the effects of GSCs plasticity on GBM progression, discussing the role of GSCs niches on modulating their phenotype. Finally, we described epigenetic alterations in GSCs that are important for stemness, cell fate and therapeutic resistance.

A novel patient stratification strategy to enhance the therapeutic efficacy of dasatinib in glioblastoma.

BACKGROUND: Glioblastoma is the most common primary malignancy of the central nervous system with a dismal prognosis. Genomic signatures classify isocitrate dehydrogenase 1 (IDH)-wildtype glioblastoma into three subtypes: proneural, mesenchymal, and classical. Dasatinib, an inhibitor of proto-oncogene kinase Src (SRC), is one of many therapeutics which, despite promising preclinical results, have failed to improve overall survival in glioblastoma patients in clinical trials. We examined whether glioblastoma subtypes differ in their response to dasatinib and could hence be evaluated for patient enrichment strategies in clinical trials. METHODS: We carried out in silico analyses on glioblastoma gene expression (TCGA) and single-cell RNA-Seq data. In addition, in vitro experiments using glioblastoma stem-like cells (GSCs) derived from primary patient tumors were performed, with complementary gene expression profiling and immunohistochemistry analysis of tumor samples. RESULTS: Patients with the mesenchymal subtype of glioblastoma showed higher SRC pathway activation based on gene expression profiling. Accordingly, mesenchymal GSCs were more sensitive to SRC inhibition by dasatinib compared to proneural and classical GSCs. Notably, SRC phosphorylation status did not predict response to dasatinib treatment. Furthermore, serpin peptidase inhibitor clade H member 1 (SERPINH1), a collagen-related heat-shock protein associated with cancer progression, was shown to correlate with dasatinib response and with the mesenchymal subtype. CONCLUSION: This work highlights further molecular-based patient selection strategies in clinical trials and suggests the mesenchymal subtype as well as SERPINH1 to be associated with response to dasatinib. Our findings indicate that stratification based on gene expression subtyping should be considered in future dasatinib trials.

Downregulated CLIP3 induces radioresistance by enhancing stemness and glycolytic flux in glioblastoma.

BACKGROUND: Glioblastoma Multiforme (GBM) is a malignant primary brain tumor in which the standard treatment, ionizing radiation (IR), achieves a median survival of about 15 months. GBM harbors glioblastoma stem-like cells (GSCs), which play a crucial role in therapeutic resistance and recurrence. METHODS: Patient-derived GSCs, GBM cell lines, intracranial GBM xenografts, and GBM sections were used to measure mRNA and protein expression and determine the related molecular mechanisms by qRT-PCR, immunoblot, immunoprecipitation, immunofluorescence, OCR, ECAR, live-cell imaging, and immunohistochemistry. Orthotopic GBM xenograft models were applied to investigate tumor inhibitory effects of glimepiride combined with radiotherapy. RESULTS: We report that GSCs that survive standard treatment radiation upregulate Speedy/RINGO cell cycle regulator family member A (Spy1) and downregulate CAP-Gly domain containing linker protein 3 (CLIP3, also known as CLIPR-59). We discovered that Spy1 activation and CLIP3 inhibition coordinately shift GBM cell glucose metabolism to favor glycolysis via two cellular processes: transcriptional regulation of CLIP3 and facilitating Glucose transporter 3 (GLUT3) trafficking to cellular membranes in GBM cells. Importantly, in combination with IR, glimepiride, an FDA-approved medication used to treat type 2 diabetes mellitus, disrupts GSCs maintenance and suppresses glycolytic activity by restoring CLIP3 function. In addition, combining radiotherapy and glimepiride significantly reduced GBM growth and improved survival in a GBM orthotopic xenograft mouse model. CONCLUSIONS: Our data suggest that radioresistant GBM cells exhibit enhanced stemness and glycolytic activity mediated by the Spy1-CLIP3 axis. Thus, glimepiride could be an attractive strategy for overcoming radioresistance and recurrence by rescuing CLIP3 expression.

Calcitriol Promotes Differentiation of Glioma Stem-Like Cells and Increases Their Susceptibility to Temozolomide.

Glioblastoma (GBM) is the most common and most aggressive primary brain tumor, with a very high rate of recurrence and a median survival of 15 months after diagnosis. Abundant evidence suggests that a certain sub-population of cancer cells harbors a stem-like phenotype and is likely responsible for disease recurrence, treatment resistance and potentially even for the infiltrative growth of GBM. GBM incidence has been negatively correlated with the serum levels of 25-hydroxy-vitamin D3, while the low pH within tumors has been shown to promote the expression of the vitamin D3-degrading enzyme 24-hydroxylase, encoded by the CYP24A1 gene. Therefore, we hypothesized that calcitriol can specifically target stem-like glioblastoma cells and induce their differentiation. Here, we show, using in vitro limiting dilution assays, quantitative real-time PCR, quantitative proteomics and ex vivo adult organotypic brain slice transplantation cultures, that therapeutic doses of calcitriol, the hormonally active form of vitamin D3, reduce stemness to varying extents in a panel of investigated GSC lines, and that it effectively hinders tumor growth of responding GSCs ex vivo. We further show that calcitriol synergizes with Temozolomide ex vivo to completely eliminate some GSC tumors. These findings indicate that calcitriol carries potential as an adjuvant therapy for a subgroup of GBM patients and should be analyzed in more detail in follow-up studies.

The potential use of tideglusib as an adjuvant radio-therapeutic treatment for glioblastoma multiforme cancer stem-like cells.

BACKGROUND: Glioblastoma multiforme (GBM), a stage IV astrocytoma, is the most common brain malignancy among adults. Conventional treatments of surgical resection followed by radio and/or chemotherapy fail to completely eradicate the tumor. Resistance to the currently available therapies is mainly attributed to a subpopulation of cancer stem cells (CSCs) present within the tumor bulk that self-renew leading to tumor relapse with time. Therefore, identification of characteristic markers specific to these cells is crucial for the development of targeted therapies. Glycogen synthase kinase 3 (GSK-3), a serine-threonine kinase, is deregulated in a wide range of diseases, including cancer. In GBM, GSK-3ß is overexpressed and its suppression in vitro has been shown to induce apoptosis of cancer cells. METHODS: In our study, we assessed the effect of GSK-3ß inhibition with Tideglusib (TDG), an irreversible non-ATP competitive inhibitor, using two human GBM cell lines, U-251 MG and U-118 MG. In addition, we combined TDG with radiotherapy to assess whether this inhibition enhances the effect of standard treatment. RESULTS: Our results showed that TDG significantly reduced cell proliferation, cell viability, and migration of both GBM cell lines in a dose- and time-dependent manner in vitro. Treatment with TDG alone and in combination with radiation significantly decreased the colony formation of U-251 MG cells and the sphere formation of both cell lines, by targeting and reducing their glioblastoma cancer stem-like cells (GSCs) population. Finally, cells treated with TDG showed an increased level of unrepaired radio-induced DNA damage and, thus, became sensitized toward radiation. CONCLUSIONS: In conclusion, TDG has proven its effectiveness in targeting the cancerous properties of GBM in vitro and may, hence, serve as a potential adjuvant radio-therapeutic agent to better target this deadly tumor.