Differentiated Glioblastoma Cells Research Articles

Advances in Anti-Cancer Drug Development: Metformin as Anti-Angiogenic Supplemental Treatment for Glioblastoma

According to the WHO 2016 classification, glioblastoma is the most prevalent primary tumor in the adult central nervous system (CNS) and is categorized as grade IV. With an average lifespan of about 15 months from diagnosis, glioblastoma has a poor prognosis and presents a significant treatment challenge. Aberrant angiogenesis, which promotes tumor neovascularization and is a prospective target for molecular target treatment, is one of its unique and aggressive characteristics. Recently, the existence of glioma stem cells (GSCs) within the tumor, which are tolerant to chemotherapy and radiation, has been linked to the highly aggressive form of glioblastoma. Anti-angiogenic medications have not significantly improved overall survival (OS), despite various preclinical investigations and clinical trials demonstrating encouraging results. This suggests the need to discover new treatment options. Glioblastoma is one of the numerous cancers for which metformin, an anti-hyperglycemic medication belonging to the Biguanides family, is used as first-line therapy for type 2 diabetes mellitus (T2DM), and it has shown both in vitro and in vivo anti-tumoral activity. Based on these findings, the medication has been repurposed, which has shown the inhibition of many oncopromoter mechanisms and, as a result, identified the molecular pathways involved. Metformin inhibits cancer cell growth by blocking the LKB1/AMPK/mTOR/S6K1 pathway, leading to selective cell death in GSCs and inhibiting the proliferation of CD133+ cells. It has minimal impact on differentiated glioblastoma cells and normal human stem cells. The systematic retrieval of information was performed on PubMed. A total of 106 articles were found in a search on metformin for glioblastoma. Out of these six articles were Meta-analyses, Randomized Controlled Trials, clinical trials, and Systematic Reviews. The rest were Literature review articles. These articles were from the years 2011 to 2024. Appropriate studies were isolated, and important information from each of them was understood and entered into a database from which the information was used in this article. The clinical trials on metformin use in the treatment of glioblastoma were searched on clinicaltrials.gov. In this article, we examine and evaluate metformin’s possible anti-tumoral effects on glioblastoma, determining whether or not it may appropriately function as an anti-angiogenic substance and be safely added to the treatment and management of glioblastoma patients.

P02.22.B CELLULAR COMPOSITION DETERMINED BY STEMNESS MARKERS IN MONOLAYER GLIOBLASTOMA CULTURES IS TUMOR SPECIFIC, HIGHLY DYNAMIC AND RELATED TO TMZ SENSITIVITY

Abstract BACKGROUND Different models are used to study pathophysiology and potential new therapeutic strategies of glioblastoma (GBM) in vitro. Generally, monolayer cultures are chosen because of their high reproducibility, while spheroid cultures are preferred to better recapitulate tumor heterogeneity and effects of the micro-environment. A common assumption is that monolayer cultures display less plasticity than spheroid cultures. In this study we examine the plasticity of stem cell-like cells in monolayer cultures and their relationship to temozolomide (TMZ) sensitivity. MATERIAL AND METHODS Three patient-derived GBM cultures: one TMZ sensitive, one induced TMZ-resistant and one naturally TMZ-resistant were used. All cultures were sorted in four subpopulations (CD133+/CD44+, CD133+/CD44-, CD133-/CD44+ and CD133-/CD44-) and subsequently cultured. Cellular composition was determined at multiple timepoints by immunofluorescent imaging and FACS-analysis. The effect of seeding density on cellular composition was assessed. Furthermore the effect of original culture composition on sphere forming capacity and TMZ sensitivity was assessed. RESULTS Culture composition was tumor-specific and the pre-sorting composition was acquired within two passages after sorting, irrespective of original subpopulation or sorting density. Even the CD133-/CD44- subpopulation was able to rapidly re-acquire the initial tumor composition. Furthermore, sphere-forming capacity was restored four passages after sorting. Interestingly, cells that were inherently resistant to TMZ expressed higher levels of CD44 than TMZ-sensitive cells. There was no relationship between expression of CD133 and sensitivity to TMZ. Sensitivity to TMZ was similar in all subpopulations four passages after sorting. CONCLUSION Monolayer cultures display rapid plasticity with regard to the expression of the stemness markers CD133 and CD44. Culture composition is tumor specific and inherently programmed in each tumor cell. TMZ sensitivity seems to be mostly related to CD44 expression, and not to CD133 expression. Sphere forming capacity and TMZ sensitivity are restored soon after sorting, even in initially differentiated GBM cells.

The Cytotoxic Effects of Cannabidiol and Cannabigerol on Glioblastoma Stem Cells May Mostly Involve GPR55 and TRPV1 Signalling.

Glioblastoma (GBM) is one of the most aggressive cancers, comprising 60-70% of all gliomas. The large G-protein-coupled receptor family includes cannabinoid receptors CB1, CB2, GPR55, and non-specific ion receptor protein transporters TRPs. First, we found up-regulated CNR1, GPR55, and TRPV1 expression in glioma patient-derived tissue samples and cell lines compared with non-malignant brain samples. CNR1 and GPR55 did not correlate with glioma grade, whereas TRPV1 negatively correlated with grade and positively correlated with longer overall survival. This suggests a tumour-suppressor role of TRPV1. With respect to markers of GBM stem cells, preferred targets of therapy, TRPV1 and GPR55, but not CNR1, strongly correlated with different sets of stemness gene markers: NOTCH, OLIG2, CD9, TRIM28, and TUFM and CD15, SOX2, OCT4, and ID1, respectively. This is in line with the higher expression of TRPV1 and GPR55 genes in GSCs compared with differentiated GBM cells. Second, in a panel of patient-derived GSCs, we found that CBG and CBD exhibited the highest cytotoxicity at a molar ratio of 3:1. We suggest that this mixture should be tested in experimental animals and clinical studies, in which currently used Δ9-tetrahydrocannabinol (THC) is replaced with efficient and non-psychoactive CBG in adjuvant standard-of-care therapy.

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.

Targeting Nuclear Pore Complex to Radiosensitize Glioblastoma Stem Cells

<h3>Purpose/Objective(s)</h3> Glioblastoma is the most prevalent and lethal primary intrinsic tumor in the central nervous system. Glioblastoma stem cells (GSCs) reprogram the transcriptional and metabolic machinery to mediate therapeutic resistance to radiation. Understanding the regulatory mechanism of GSCs may inform novel approaches to improve the clinical outcome for glioblastoma patients. Nuclear pore complex (NPC) is an evolutionarily conserved structure that maintains the traffic between the nucleus and cytoplasm. In current study, we aim to interrogate the aberrant regulation of NPC in GSCs and develop novel targeted approach to sensitize GSC to radiation. <h3>Materials/Methods</h3> Patient-derived GSCs were validated using functional assays. Transcriptional analysis of GSCs and matched differentiated glioblastoma cells (DGCs) were performed using RNA sequencing to identify aberrant regulatory pathways upregulated in GSCs. The clinical impact of target genes on patient survival was assessed using the glioblastoma dataset from The Cancer Genome Atlas (TCGA). Quantitative RT-PCR and Western blotting were performed to assess the relative expression between GSCs and DGCs. Radiation sensitivity was measured using Cs-137 irradiator. Validation of the stem-like function of target genes was performed using RNA interference followed by <i>in vitro</i> neurosphere formation assay and <i>in vivo</i> tumorigenesis with xenograft mouse model. <h3>Results</h3> 27 of the total 31 NPC genes were found to be upregulated in the primary glioblastoma patient specimens compared to non-tumor brain tissue in the TCGA dataset. Among them, a systematic approach combining transcriptional profiling and clinical validation linked nucleoporin 98 (NUP98) to a nexus between GSC phenotype and radiation resistance. GSCs preferentially express NUP98, which is essential for GSC maintenance and tumorigenesis <i>in vitro</i> and <i>in vivo</i>. RNA sequencing demonstrated that NUP98 regulates the expression of key DNA repair genes, including BRCA1, BRCA2, and RAD51. NUP98 forms a complex with the transcription factor P65, and directly stimulates the active transcription of genes involved in homologous recombination. Attenuation of NUP98 or P65 expression leads to the accumulation of unrepaired intrinsic DNA damage and sensitizes GSC to the treatment of ionizing radiation. Clinically, overexpression of NUP98 informs poor clinical outcome among patients with glioblastoma. <h3>Conclusion</h3> Combining transcriptional discovery and clinical validation, we discovered that NUP98, an essential NPC component, maintains GSC proliferation and tumorigenesis through modulating the transcriptional control of homologous recombination pathway. Our results demonstrated a novel therapeutic approach of sensitizing GSC to radiation through targeting the nexus between aberrant transcriptional regulation and therapeutic resistance in stem-like glioma cells.

Targeting nuclear pore complex and therapeutic response in glioblastoma stem cells.

e14000 Background: Glioblastoma is the most prevalent and lethal primary intrinsic tumor in the central nervous system. Glioblastoma stem cells (GSCs) reprogram the transcriptional and metabolic machinery to survive in a dynamic microenvironment. Understanding the regulatory mechanism of GSCs may inform novel therapeutic approaches to improve the clinical outcome for glioblastoma patients. Nuclear pore complex (NPC) is an evolutionarily conserved structure that maintains the traffic between the nucleus and cytoplasm. In current study, we aim to interrogate the aberrant regulation of NPC in GSCs and develop novel targeted approach to sensitize GSC to conventional therapy. Methods: Patient-derived GSCs were validated using functional assays. Transcriptional analysis of GSCs and matched differentiated glioblastoma cells (DGCs) were performed using RNA sequencing to identify aberrant regulatory pathways upregulated in GSCs. The clinical impact of target genes on patient survival was assessed using the glioblastoma dataset from The Cancer Genome Atlas (TCGA). Quantitative RT-PCR and Western blotting were performed to assess the relative expression between GSCs and DGCs. Radiation sensitivity was measured using Cs-137 irradiator. Validation of the stem-like function of target genes was performed using RNA interference followed by in vitro neurosphere formation assay and in vivo tumorigenesis with xenograft mouse model. Results: 27 of the total 31 NPC genes were found to be upregulated in the primary glioblastoma patient specimens compared to non-tumor brain tissue in the TCGA dataset. Among them, a systemic approach combining transcriptional profiling and clinical validation linked nucleoporin 98 (NUP98) to a nexus between GSC phenotype and therapeutic resistance. GSCs preferentially express NUP98, which is essential for GSC maintenance and tumorigenesis in vitro and in vivo. RNA sequencing demonstrated that NUP98 regulates the expression of key DNA repair genes, including BRCA1, BRCA2, and RAD51. NUP98 forms a complex with the transcription factor P65, and directly stimulates the active transcription of genes involved in homologous recombination. Attenuation of NUP98 or P65 expression leads to the accumulation of unrepaired intrinsic DNA damage and sensitizes GSC to the treatment of ionizing radiation. Clinically, overexpression of NUP98 informs poor clinical outcome among patients with glioblastoma. Conclusions: Combining transcriptional discovery and clinical validation, we discovered that NUP98, an essential NPC component, maintains GSC proliferation and tumorigenesis through modulating the transcriptional control of homologous recombination pathway. Our results demonstrated a novel therapeutic approach of sensitizing GSC to conventional treatment through targeting the nexus between aberrant transcriptional regulation and therapeutic resistance in stem-like glioma cells.

In This Issue

In This Issue

Transcription Elongation Machinery Is a Druggable Dependency and Potentiates Immunotherapy in Glioblastoma Stem Cells.

Glioblastoma (GBM) is the most lethal primary brain cancer characterized by therapeutic resistance, which is promoted by GBM stem cells (GSC). Here, we interrogated gene expression and whole-genome CRISPR/Cas9 screening in a large panel of patient-derived GSCs, differentiated GBM cells (DGC), and neural stem cells (NSC) to identify master regulators of GSC stemness, revealing an essential transcription state with increased RNA polymerase II-mediated transcription. The YY1 and transcriptional CDK9 complex was essential for GSC survival and maintenance in vitro and in vivo. YY1 interacted with CDK9 to regulate transcription elongation in GSCs. Genetic or pharmacologic targeting of the YY1-CDK9 complex elicited RNA m6A modification-dependent interferon responses, reduced regulatory T-cell infiltration, and augmented efficacy of immune checkpoint therapy in GBM. Collectively, these results suggest that YY1-CDK9 transcription elongation complex defines a targetable cell state with active transcription, suppressed interferon responses, and immunotherapy resistance in GBM. SIGNIFICANCE: Effective strategies to rewire immunosuppressive microenvironment and enhance immunotherapy response are still lacking in GBM. YY1-driven transcriptional elongation machinery represents a druggable target to activate interferon response and enhance anti-PD-1 response through regulating the m6A modification program, linking epigenetic regulation to immunomodulatory function in GBM.This article is highlighted in the In This Issue feature, p. 275.

Can ECIS Biosensor Technology Be Used to Measure the Cellular Responses of Glioblastoma Stem Cells?

Glioblastoma is considered the most aggressive and lethal form of brain cancer. Glioblastoma tumours are complex, comprising a spectrum of oncogenically transformed cells displaying distinct phenotypes. These can be generated in culture and are called differentiated-glioblastoma cells and glioblastoma stem cells. These cells are phenotypically and functionally distinct, where the stem-like glioblastoma cells give rise to and perpetuate the tumour. Electric cell-substrate impedance sensing (ECIS) is a real-time, label-free, impedance-based method for the analysis of cellular behaviour, based on cellular adhesion. Therefore, we asked the question of whether ECIS was suitable for, and capable of measuring the adhesion of glioblastoma cells. The goal was to identify whether ECIS was capable of measuring glioblastoma cell adhesion, with a particular focus on the glioblastoma stem cells. We reveal that ECIS reliably measures adhesion of the differentiated glioblastoma cells on various array types. We also demonstrate the ability of ECIS to measure the migratory behaviour of differentiated glioblastoma cells onto ECIS electrodes post-ablation. Although the glioblastoma stem cells are adherent, ECIS is substantially less capable at reliably measuring their adhesion, compared with the differentiated counterparts. This means that ECIS has applicability for some glioblastoma cultures but much less utility for weakly adherent stem cell counterparts.

Targeting EYA2 tyrosine phosphatase activity in glioblastoma stem cells induces mitotic catastrophe.

Glioblastoma ranks among the most lethal of primary brain malignancies, with glioblastoma stem cells (GSCs) at the apex of tumor cellular hierarchies. Here, to discover novel therapeutic GSC targets, we interrogated gene expression profiles from GSCs, differentiated glioblastoma cells (DGCs), and neural stem cells (NSCs), revealing EYA2 as preferentially expressed by GSCs. Targeting EYA2 impaired GSC maintenance and induced cell cycle arrest, apoptosis, and loss of self-renewal. EYA2 displayed novel localization to centrosomes in GSCs, and EYA2 tyrosine (Tyr) phosphatase activity was essential for proper mitotic spindle assembly and survival of GSCs. Inhibition of the EYA2 Tyr phosphatase activity, via genetic or pharmacological means, mimicked EYA2 loss in GSCs in vitro and extended the survival of tumor-bearing mice. Supporting the clinical relevance of these findings, EYA2 portends poor patient prognosis in glioblastoma. Collectively, our data indicate that EYA2 phosphatase function plays selective critical roles in the growth and survival of GSCs, potentially offering a high therapeutic index for EYA2 inhibitors.

TRIM28 Selective Nanobody Reduces Glioblastoma Stem Cell Invasion.

Glioblastoma (GB), is the most common and aggressive malignant primary brain tumour in adults. Intra- and inter-tumour heterogeneity, infiltrative GB cell invasion and presence of therapy-resistant GB stem cells (GSCs) represent major obstacles to favourable prognosis and poor therapy response. Identifying the biomarkers of the most aggressive tumour cells and their more efficient targeting strategies are; therefore, crucial. Recently, transcription factor TRIM28 has been identified as a GB biomarker and, in this study, we have shown high expression of TRIM28 in GB and in low grade gliomas as well as higher expression in GSCs vs. differentiated GB cells, although in both cases not significant. We demonstrated significant in vitro inhibition of GB cells and GSCs invasiveness and spread in zebrafish brains in vivo by anti-TRIM28 selective nanobody NB237. TRIM28 was also enriched in GB (tumour) core and associated with the expression of stem cell genes, but was not prognostic for overall survival. However, based on the above results, we conclude that TRIM28 nanobody NB237 offers a new opportunity as a GB therapeutic tool.

Investigation of the lipidomic changes in differentiated glioblastoma cells after drug treatment using MALDI-MS

Lipids differences between tumor and normal tissue have been proved to be of diagnostic and therapeutic significance. The research of lipidomics in tumor is more and more important. Mass spectrometry like matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) can be more convenient and informative for lipids researching in biological and clinical researches. Most of malignant tumors like glioblastoma are characterized by incomplete differentiation, so differentiation therapy has made important progress in tumor treatment. Lipid profiles changes after therapy are worthy investigating. In our study, glioblastoma cell line U87-MG cells were treated by inducers of sodium phenylbutyrate (SPB) and all-trans retinoic acid (ATRA). The changes in lipids on cell membrane were profiled by MALDI-MS. The differentiation degree was assessed by cell proliferation, cell cycle, morphology and protein expression before MALDI-MS analysis. Comparing the inducer treated and untreated U87-MG cells, reduced proliferation rate, blocked cell cycle, benign nucleus morphology and changed expression of protein CD133 and glial fibrillary acidic protein (GFAP), were found after drug treatment. Moreover, the lipids of cell membrane presented distinguished differences in the drug treated cells. Most of the glycerophosphocholines (PC) with an increasing abundance are unsaturated PCs (PC (38:1), 816 m/z; PC (36:1), 788 m/z; PC (31:1), 725 m/z), and those decreasing are saturated PCs (PC (32:0), 734 m/z). These results provide the lipidomic differentiation which may be a significant guidance for evaluating the therapeutic effect of tumor therapy.

Energy Metabolism in IDH1 Wild-Type and IDH1-Mutated Glioblastoma Stem Cells: A Novel Target for Therapy?

Cancer is a redox disease. Low levels of reactive oxygen species (ROS) are beneficial for cells and have anti-cancer effects. ROS are produced in the mitochondria during ATP production by oxidative phosphorylation (OXPHOS). In the present review, we describe ATP production in primary brain tumors, glioblastoma, in relation to ROS production. Differentiated glioblastoma cells mainly use glycolysis for ATP production (aerobic glycolysis) without ROS production, whereas glioblastoma stem cells (GSCs) in hypoxic periarteriolar niches use OXPHOS for ATP and ROS production, which is modest because of the hypoxia and quiescence of GSCs. In a significant proportion of glioblastoma, isocitrate dehydrogenase 1 (IDH1) is mutated, causing metabolic rewiring, and all cancer cells use OXPHOS for ATP and ROS production. Systemic therapeutic inhibition of glycolysis is not an option as clinical trials have shown ineffectiveness or unwanted side effects. We argue that systemic therapeutic inhibition of OXPHOS is not an option either because the anti-cancer effects of ROS production in healthy cells is inhibited as well. Therefore, we advocate to remove GSCs out of their hypoxic niches by the inhibition of their binding to niches to enable their differentiation and thus increase their sensitivity to radiotherapy and/or chemotherapy.

Differentiated glioblastoma cells accelerate tumor progression by shaping the tumor microenvironment via CCN1-mediated macrophage infiltration

Glioblastoma (GBM) is the most lethal primary brain tumor characterized by significant cellular heterogeneity, namely tumor cells, including GBM stem-like cells (GSCs) and differentiated GBM cells (DGCs), and non-tumor cells such as endothelial cells, vascular pericytes, macrophages, and other types of immune cells. GSCs are essential to drive tumor progression, whereas the biological roles of DGCs are largely unknown. In this study, we focused on the roles of DGCs in the tumor microenvironment. To this end, we extracted DGC-specific signature genes from transcriptomic profiles of matched pairs of in vitro GSC and DGC models. By evaluating the DGC signature using single cell data, we confirmed the presence of cell subpopulations emulated by in vitro culture models within a primary tumor. The DGC signature was correlated with the mesenchymal subtype and a poor prognosis in large GBM cohorts such as The Cancer Genome Atlas and Ivy Glioblastoma Atlas Project. In silico signaling pathway analysis suggested a role of DGCs in macrophage infiltration. Consistent with in silico findings, in vitro DGC models promoted macrophage migration. In vivo, coimplantation of DGCs and GSCs reduced the survival of tumor xenograft-bearing mice and increased macrophage infiltration into tumor tissue compared with transplantation of GSCs alone. DGCs exhibited a significant increase in YAP/TAZ/TEAD activity compared with GSCs. CCN1, a transcriptional target of YAP/TAZ, was selected from the DGC signature as a candidate secreted protein involved in macrophage recruitment. In fact, CCN1 was secreted abundantly from DGCs, but not GSCs. DGCs promoted macrophage migration in vitro and macrophage infiltration into tumor tissue in vivo through secretion of CCN1. Collectively, these results demonstrate that DGCs contribute to GSC-dependent tumor progression by shaping a mesenchymal microenvironment via CCN1-mediated macrophage infiltration. This study provides new insight into the complex GBM microenvironment consisting of heterogeneous cells.

Mitochondria's Role in the Maintenance of Cancer Stem Cells in Glioblastoma.

Glioblastoma (GBM), one of the deadliest primary brain malignancies, is characterized by a high recurrence rate due to its limited response to existing therapeutic strategies such as chemotherapy, radiation therapy, and surgery. Several mechanisms and pathways have been identified to be responsible for GBM therapeutic resistance. Glioblastoma stem cells (GSCs) are known culprits of GBM resistance to therapy. GSCs are characterized by their unique self-renewal, differentiating capacity, and proliferative potential. They form a heterogeneous population of cancer stem cells within the tumor and are further divided into different subpopulations. Their distinct molecular, genetic, dynamic, and metabolic features distinguish them from neural stem cells (NSCs) and differentiated GBM cells. Novel therapeutic strategies targeting GSCs could effectively reduce the tumor-initiating potential, hence, a thorough understanding of mechanisms involved in maintaining GSCs’ stemness cannot be overemphasized. The mitochondrion, a regulator of cellular physiological processes such as autophagy, cellular respiration, reactive oxygen species (ROS) generation, apoptosis, DNA repair, and cell cycle control, has been implicated in various malignancies (for instance, breast, lung, and prostate cancer). Besides, the role of mitochondria in GBM has been extensively studied. For example, when stressors, such as irradiation and hypoxia are present, GSCs utilize specific cytoprotective mechanisms like the activation of mitochondrial stress pathways to survive the harsh environment. Proliferating GBM cells exhibit increased cytoplasmic glycolysis in comparison to terminally differentiated GBM cells and quiescent GSCs that rely more on oxidative phosphorylation (OXPHOS). Furthermore, the Warburg effect, which is characterized by increased tumor cell glycolysis and decreased mitochondrial metabolism in the presence of oxygen, has been observed in GBM. Herein, we highlight the importance of mitochondria in the maintenance of GSCs.

Integrative Analysis of miRNA-mediated Competing Endogenous RNA Network Reveals the lncRNAs-mRNAs Interaction in Glioblastoma Stem Cell Differentiation

Background: Competing endogenous RNA (ceRNA) networks play a pivotal role in tumor diagnosis and progression. Numerous studies have explored the functional landscape and prognostic significance of ceRNA interaction within differentiated tumor cells. Objective: We propose a new perspective by exploring ceRNA networks in the process of glioblastoma stem cell (GSC) differentiation. Methods: In this study, expression profiles of lncRNAs and mRNAs were compared between GSCs and differentiated glioblastoma cells. Using a comprehensive computational method, miRNAmediated and GSC differentiation-associated ceRNA crosstalk between lncRNAs and mRNAs was identified. A ceRNA network was then established to select potential candidates that regulate GSC differentiation. Results: Based on the specific ceRNA network related to GSC differentiation, we identified lnc MYOSLID: 11 as a ceRNA that regulated the expression of the downstream gene PXN by competitively binding with hsa-miR-149-3p. After Kaplan-Meier (KM) survival analysis, the expression of PXN gene (PPXN = 0.0015) and lnc MYOSLID: 11 (PMYOSLID: 11=0.041) showed significant correlation with glioblastoma in 160 patients from TCGA. Conclusion: This result sheds light on a potential way of studying the ceRNA network, which can provide clues for developing new diagnostic methods and finding therapeutic targets for clinical treatment of glioblastoma.

Single-cell analyses reveal YAP/TAZ as regulators of stemness and cell plasticity in Glioblastoma.

Glioblastoma (GBM) is a devastating human malignancy. GBM stem-like cells (GSCs) drive tumor initiation and progression. Yet, the molecular determinants defining GSCs in their native state in patients remain poorly understood. Here we used single cell datasets and identified GSCs at the apex of the differentiation hierarchy of GBM. By reconstructing the GSCs’ regulatory network, we identified the YAP/TAZ coactivators as master regulators of this cell state, irrespectively of GBM subtypes. YAP/TAZ are required to install GSC properties in primary cells downstream of multiple oncogenic lesions, and required for tumor initiation and maintenance in vivo in different mouse and human GBM models. YAP/TAZ act as main roadblock of GSC differentiation and their inhibition irreversibly lock differentiated GBM cells into a non-tumorigenic state, preventing plasticity and regeneration of GSC-like cells. Thus, GSC identity is linked to a key molecular hub integrating genetics and microenvironmental inputs within the multifaceted biology of GBM.

The HIF1\u03b1/HIF2\u03b1-miR210-3p network regulates glioblastoma cell proliferation, dedifferentiation and chemoresistance through EGF under hypoxic conditions

Hypoxia-inducible factor 1α (HIF1α) promotes the malignant progression of glioblastoma under hypoxic conditions, leading to a poor prognosis for patients with glioblastoma; however, none of the therapies targeting HIF1α in glioblastoma have successfully eradicated the tumour. Therefore, we focused on the reason and found that treatments targeting HIF1α and HIF2α simultaneously increased tumour volume, but the combination of HIF1α/HIF2α-targeted therapies with temozolomide (TMZ) reduced tumourigenesis and significantly improved chemosensitization. Moreover, miR-210-3p induced HIF1α expression but inhibited HIF2α expression, suggesting that miR-210-3p regulates HIF1α/HIF2α expression. Epidermal growth factor (EGF) has been shown to upregulate HIF1α expression under hypoxic conditions. However, in the present study, in addition to the signalling pathways mentioned above, the upstream proteins HIF1α and HIF2α have been shown to induce EGF expression by binding to the sequences AGGCGTGG and GGGCGTGG. Briefly, in a hypoxic microenvironment the HIF1α/HIF2α-miR210-3p network promotes the malignant progression of glioblastoma through a positive feedback loop with EGF. Additionally, differentiated glioblastoma cells underwent dedifferentiation to produce glioma stem cells under hypoxic conditions, and simultaneous knockout of HIF1α and HIF2α inhibited cell cycle arrest but promoted proliferation with decreased stemness, promoting glioblastoma cell chemosensitization. In summary, both HIF1α and HIF2α regulate glioblastoma cell proliferation, dedifferentiation and chemoresistance through a specific pathway, which is important for glioblastoma treatments.

Temozolomide Treatment Increases Fatty Acid Uptake in Glioblastoma Stem Cells.

Simple SummaryPatients diagnosed with glioblastoma (GBM) brain tumors typically survive less than two years, despite aggressive therapy with surgery, radiation, and chemotherapy. A major factor underlying this lethality is the ability of GBM tumors to adapt to stress, including the stress of treatment. The role of metabolism in this process remains incompletely understood. We, therefore, explored the connection between cellular phenotype, chemotherapeutic stress, and metabolism in GBM. We found that inducing changes in GBM phenotypes led to alterations in metabolic behavior. Further, during treatment with chemotherapy, GBM cells that became resistant to therapy increased their fatty acid uptake. These therapy-induced alterations in nutrient uptake may underlie therapy resistance and deadly recurrence.Among all cancers, glioblastoma (GBM) remains one of the least treatable. One key factor in this resistance is a subpopulation of tumor cells termed glioma stem cells (GSCs). These cells are highly resistant to current treatment modalities, possess marked self-renewal capacity, and are considered key drivers of tumor recurrence. Further complicating an understanding of GBM, evidence shows that the GSC population is not a pre-ordained and static group of cells but also includes previously differentiated GBM cells that have attained a GSC state secondary to environmental cues. The metabolic behavior of GBM cells undergoing plasticity remains incompletely understood. To that end, we probed the connection between GSCs, environmental cues, and metabolism. Using patient-derived xenograft cells, mouse models, transcriptomics, and metabolic analyses, we found that cell state changes are accompanied by sharp changes in metabolic phenotype. Further, treatment with temozolomide, the current standard of care drug for GBM, altered the metabolism of GBM cells and increased fatty acid uptake both in vitro and in vivo in the plasticity driven GSC population. These results indicate that temozolomide-induced changes in cell state are accompanied by metabolic shifts—a potentially novel target for enhancing the effectiveness of current treatment modalities.

A computational guided, functional validation of a novel therapeutic antibody proposes Notch signaling as a clinical relevant and druggable target in glioma

The Notch signaling network determines stemness in various tissues and targeting signaling activity in malignant brain cancers by gamma-secretase inhibitors (GSI) has shown promising preclinical success. However, the clinical translation remains challenging due to severe toxicity side effects and emergence of therapy resistance. Better anti-Notch directed therapies, specifically directed against the tumor promoting Notch receptor 1 signaling framework, and biomarkers predicting response to such therapy are of highest clinical need. We assessed multiple patient datasets to probe the clinical relevance Notch1 activation and possible differential distribution amongst molecular subtypes in brain cancers. We functionally assessed the biological effects of the first-in-human tested blocking antibody against Notch1 receptor (brontictuzumab, BRON) in a collection of glioma stem-like cell (GSC) models and compared its effects to genetic Notch1 inhibition as well as classical pharmacological Notch inhibitor treatment using gamma-secretase inhibitor MRK003. We also assess effects on Wingless (WNT) stem cell signaling activation, which includes the interrogation of genetic WNT inhibition models. Our computed transcriptional Notch pathway activation score is upregulated in neural stem cells, as compared to astrocytes; as well as in GSCs, as compared to differentiated glioblastoma cells. Moreover, the Notch signature is clinical predictive in our glioblastoma patient discovery and validation cohort. Notch signature is significantly increased in tumors with mutant IDH1 genome and tumors without 1p and 19q co-deletion. In GSCs with elevated Notch1 expression, BRON treatment blocks transcription of Notch pathway target genes Hes1/Hey1, significantly reduced the amount of cleaved Notch1 receptor protein and caused significantly impairment of cellular invasion. Benchmarking this phenotype to those observed with genetic Notch1 inhibition in corresponding cell models did result in higher reduction of cell invasion under chemotherapy. BRON treatment caused signs of upregulation of Wingless (WNT) stem cell signaling activity, and vice versa, blockage of WNT signaling caused induction of Notch target gene expression in our models. We extend the list of evidences that elevated Notch signal expression is a biomarker signature declaring stem cell prevalence and useful for predicting negative clinical course in glioblastoma. By using functional assays, we validated a first in man tested Notch1 receptor specific antibody as a promising drug candidate in the context of neuro oncology and propose biomarker panel to predict resistance and therapy success of this treatment option. We note that the observed phenotype seems only in part due to Notch1 blockage and the drug candidate leads to activation of off target signals. Further studies addressing a possible emergence of therapy resistance due to WNT activation need to be conducted. We further validated our 3D disease modeling technology to be of benefit for drug development projects.