Glioma Cell Survival Research Articles

TMIC-20. SERINE AND ONE-CARBON METABOLISM ADAPTING GLIOMA CELLS TO LOW GLUTAMINE MICROENVIRONMENT

Abstract Cancer cells optimize nutrient utilization to supply energetic and biosynthetic pathways. This metabolic process also includes redox maintenance and epigenetic regulation through nucleic acid and protein methylation, which enhance tumorigenicity and clinical resistance. However, less is known about how cancer cells exhibit metabolic flexibility to sustain cell growth and survival from low nutrient microenvironment. Here, we find that serine and glycine levels were higher in low-nutrient regions of tumors in glioblastoma multiforme (GBM) patients than they were in other regions. Metabolic and functional studies in malignant glioma cells demonstrated that serine availability and one-carbon metabolism support glioma cell survival following glutamine deprivation. Serine synthesis was mediated through autophagy rather than glycolysis. Gene expression analysis identified upregulation of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) to regulate one-carbon metabolism in malignant glioma cells and GBM patient-derived sphere cells. In clinical samples, MTHFD2 expression was highest in the nutrient-poor areas around “pseudopalisading necrosis.” Genetic and pharmacological suppression of MTHFD2 caused tumor cell death and growth inhibition of glioma cells upon glutamine deprivation as well as autophagy inhibition did. These results may have important implications for serine-dependent one-carbon metabolism for glioma cells to survive glutamine starvation and suggest a new therapeutic strategy for patients with malignant glioma.

EXTH-67. PRECLINICAL ASSESSMENT OF THE EFFICACY OF MPT0B640, AN ORALLY BIOAVAILABLE SMALL MOLECULE HSP90 INHIBITOR, AGAINST GLIOMAS

Abstract BACKGROUND Heat-shock protein 90 (HSP90), an evolutionary conserved protein, is a key member of the epichaperome complex involved in maintenance of protein homeostasis during non-stress and stress conditions. It is overexpressed in gliomas and supports tumor cell survival and resistance. Hsp90 inhibition can impact several pathways essential for glioma cell survival and overcome tumor heterogeneity. Here, we examined the activity and toxicity profile of MPT0B640, an orally bioavailable Hsp90 inhibitor, against gliomas and its ability to cross the blood-brain barrier (BBB). METHODS Using genetically heterogeneous glioma stem-like cell (GSC) lines, we determined the effects of MPT0B640 on cell proliferation (Cell Titre Glo assay), apoptosis induction and cell cycle changes (flowcytometry; AnnexinV/PI staining), and alterations in protein expressions (western blot and reverse-phase protein array, RPPA assays). Pharmacokinetic and pharmacodynamic (PK/PD) studies were performed in non-tumor bearing mice to assess BBB permeability and antitumor effects of MPT0B640. RESULTS MPT0B640 inhibited proliferation of both adherent (LN229, A172, U251-HF) and patient-derived GSCs (GSC262, GSC811, GSC11, GSC23, GSC20 and GSC272) in a dose- and time-dependent fashion with an IC50 range of 50-1000 nM. MPT0B640-treated cells underwent apoptosis and/or cell cycle arrest with downregulation of pro-survival signal kinases, phospho-Akt, p-ERK1/2 and p-S6. PK/PD studies showed increased HSP70 expression (a marker of Hsp90 inhibition) and detectable levels of drug in brain specimens from non-tumor bearing mice after a single dose of MPT0B640, indicating BBB permeability and target inhibition of the agent. Results of ongoing animal studies assessing drug toxicity and animal survival with MPT0B640 will be presented. CONCLUSIONS MPT0B640 is a potent HSP90 inhibitor that has potent antitumor activity against genetically heterogeneous gliomas in vitro, effectively crosses the intact BBB and demonstrates target inhibition warranting further investigation for treatment of gliomas.

The role of angiogenic factors eNOS / VEGF in the treatment of anaplastic glioma

Introduction: Anaplastic gliomas (AG) account for 6–15 % of all primary brain tumors. These include: anaplastic astrocytoma (AA), anaplastic oligodendroglioma (AO), anaplastic oligoastrocytoma (AOA), and rarer forms — anaplastic pleomorphic xanthoastrocytoma and anaplastic ganglioma. According to the data on these factors, endothelial nitric oxide synthase (eNOS) is promising in terms of the prognostic value of the course of the tumor process. It was reported that a number of vascular VEGF factors interact with eNOS, contributing to the formation of an intra-tumor vascular network, which can create conditions for uneven prolonged hypoxia, leading to the emergence of more stable tumor cells. Its role in the development of a higher anaplasia level has not been studied in isolation, which determines the relevance of this study. The prognostic role of changes in endothelial nitric oxide synthase (eNOS) in the continued growth and malignant transformation of anaplastic gliomas was studied. Results: Histological samples of brain tumors of 22 patients at the University Clinic in Nizhny Novgorod from 2017 to 2019 were examined and verified for the presence of high-grade III glioma, according to the data of the World Health Organization. The average age of the patients was 50.7 years. The material was obtained as a result of surgical removal of recurrent tumors after chemo and radiotherapy. Discussion: The microenvironment of anaplastic glioma plays an essential role in its pathogenesis. More importantly, angiogenesis, which causes the supply of glioma cells with oxygen, growth factors, nutrients, and hormones, is a significant process of tumor dissemination and growth. The degree of microvascular proliferation and angiogenesis was associated with poor survival rate, transition from a lower grade to a high grade, and relapse. In high-grade glioma, such as anaplastic glioma, neoangiogenesis is an important physiological process that provides adequate blood supply for the proliferation, survival, and invasion of glioma cells. Conclusion: The high mortality rate in gliomas underscores the urgent need for effective treatment. The glioma pathogenesis is complex and can be caused by various mechanisms, as evidenced by abnormal activation of tumor angiogenesis and mutation of isocitrate dehydrogenase. VEGF acts as a regulator of angiogenesis and is widely recognized as a critical factor in glioma development and progression. Our results suggest that VEGF and eNOS inhibition may be an effective way to control and/or block endothelial barrier damage and prevent tumor progression.

EID3 Promotes Glioma Cell Proliferation and Survival by Inactivating AMPKα1.

EID3 (EP300-interacting inhibitor of differentiation) was identified as a novel member of EID family and plays a pivotal role in colorectal cancer development. However, its role in glioma remained elusive. In current study, we identified EID3 as a novel oncogenic molecule in human glioma and is critical for glioma cell survival, proliferation and invasion. A total of five patients with glioma were recruited in present study and fresh glioma samples were removed from patients. Four weeks old male non-obese diabetic severe combined immune deficiency (NOD/SCID) mice were used as transplant recipient models. The subcutaneous tumor size was calculated and recorded every week with vernier caliper. EID3 and AMP-activated protein kinase α1 (AMPKα1) expression levels were confirmed by real-time polymerase chain reaction and Western blot assays. Colony formation assays were performed to evaluate cell proliferation. Methyl thiazolyl tetrazolium (MTT) assays were performed for cell viability assessment. Trypan blue staining approach was applied for cell death assessment. Cell Apoptosis DNA ELISA Detection Kit was used for apoptosis assessment. EID3 was preferentially expressed in glioma tissues/cells, while undetectable in astrocytes, neuronal cells, or normal brain tissues. EID3 knocking down significantly hindered glioma cell proliferation and invasion, as well as induced reduction of cell viability, apoptosis and cell death. EID3 knocking down also greatly inhibited tumor growth in SCID mice. Knocking down of AMPKα1 could effectively rescue glioma cells from apoptosis and cell death caused by EID3 absence, indicating that AMPKα1 acted as a key downstream regulator of EID3 and mediated suppression effects caused by EID3 knocking down inhibition. These findings were confirmed in glioma cells generated patient-derived xenograft models. AMPKα1 protein levels were affected by MG132 treatment in glioma, which suggested EID3 might down regulate AMPKα1 through protein degradation. Collectively, our study demonstrated that EID3 promoted glioma cell proliferation and survival by inhibiting AMPKα1 expression. Targeting EID3 might represent a promising strategy for treating glioma.

Inhibition of mTOR signaling protects human glioma cells from hypoxia-induced cell death in an autophagy-independent manner

Although malignant gliomas frequently show aberrant activation of the mammalian target of rapamycin (mTOR), mTOR inhibitors have performed poorly in clinical trials. Besides regulating cell growth and translation, mTOR controls the initiation of autophagy. By recycling cellular components, autophagy can mobilize energy resources, and has thus been attributed cancer-promoting effects. Here, we asked whether the activation of autophagy represents an escape mechanism to pharmacological mTOR inhibition in glioma cells, and explored co-treatment with mTOR and autophagy inhibitors as a therapeutic strategy. Mimicking conditions of the glioma microenvironment, glioma cells were exposed to nutrient starvation and hypoxia. We analyzed autophagic activity, cell growth, viability and oxygen consumption following (co-)treatment with the mTOR inhibitors torin2 or rapamycin, and autophagy inhibitors bafilomycin A1 or MRT68921. Changes in global proteome were quantified by mass spectrometry. In the context of hypoxia and starvation, autophagy was strongly induced in glioma cells and further increased by mTOR inhibition. While torin2 enhanced glioma cell survival, co-treatment with torin2 and bafilomycin A1 failed to promote cell death. Importantly, treatment with bafilomycin A1 alone also protected glioma cells from cell death. Mechanistically, both compounds significantly reduced cell growth and oxygen consumption. Quantitative proteomics analysis showed that bafilomycin A1 induced broad changes in the cellular proteome. More specifically, proteins downregulated by bafilomycin A1 were associated with the mitochondrial respiratory chain and ATP synthesis. Taken together, our results show that activation of autophagy does not account for the cytoprotective effects of mTOR inhibition in our in vitro model of the glioma microenvironment. Our proteomic findings suggest that the pharmacological inhibition of autophagy induces extensive changes in the cellular proteome that can support glioma cell survival under nutrient-deplete and hypoxic conditions. These findings provide a novel perspective on the complex role of autophagy in gliomas.

Determination of Antiproliferative Effects of Exopolysaccharides from Six Mushroom Species on Glioma Cells

Glial tumors are the largest and danger group of central nervous system tumors. The use of natural products now has been contemplated of exceptional value in the control of cancer. Mushrooms have been used for many centuries, not just as a food, but also to treat many illnesses. Therefore, we aimed to evaluate the effect of exopolysaccharides (EPS) obtained from six different edible mushrooms on the survival of glioma cells. In this study the effects of 0.4, 1, 2, 4 and 6 μg/mL doses of EPSs from six mushroom species Coprinus comatus, Fistulina hepatica, Panus neostrigosus, Laetiporus sulphureus, Polyporus squamosus, and Lenzites betulinus were investigated on the rat glioma cell line (C6) in two different periods by MTT assay. According to our results 0.4 and 1 μg/mL of EPSs from six mushroom species were not effective or less effective, but 2, 4 and 6 μg/mL doses killed glioma cells about 27 to 71 % for 24 hours, 35 to 78 % for 48 hours As a result, these mushroom EPSs showed different cytotoxicity to glioma cells time and dose-dependently. These findings can be suggested that the anti-tumor effects of EPSs can be potential use in clinical applications to treat glioma. Further studies are needed to understand these effects more clearly on glioma.

P12.15.B Astrocyte immunometabolic regulation of the glioblastoma microenvironment drives tumor pathogenicity

Abstract Background Malignant brain tumors are the cause of a disproportionate level of morbidity and mortality among cancer patients, an unfortunate statistic that has remained constant for decades. Despite considerable advances in the molecular characterization of these tumors, targeting the cancer cells has yet to produce significant advances in treatment. An alternative strategy is to target cells in the glioblastoma microenvironment, such as tumor associated astrocytes. Astrocytes control multiple processes in health and disease, ranging from maintaining the brain's metabolic homeostasis, to modulating neuroinflammation. However, their role in glioblastoma pathogenicity is not well understood. Material and Methods Immunocompetent mice were implanted with murine glioma cell lines and the role of astrocyte in the tumor pathogenicity was analyzed, and further investigated using in-vitro co-cultures. Results Here we report that depletion of reactive astrocytes regresses glioblastoma and prolongs mouse survival. Analysis of the tumor-associated astrocyte translatome, revealed that astrocytes initiate transcriptional programs that shape the immune and metabolic compartments in the glioma microenvironment. Specifically, their expression of CCL2 and CSF1 governs the recruitment of tumor-associated macrophages and promotes a pro-tumorigenic macrophage phenotype. Concomitantly, we demonstrate that astrocyte-derived cholesterol is key to glioma cell survival, and that targeting astrocytic cholesterol efflux, via ABCA1, halts tumor progression. In summary, astrocytes control glioblastoma pathogenicity by reprogramming the immunological properties of the tumor microenvironment and supporting the non-oncogenic metabolic dependency of glioblastoma on cholesterol. Conclusion These findings suggest that targeting astrocyte immunometabolic signaling may help treat this uniformly lethal brain tumor.

Astrocyte immunometabolic regulation of the tumour microenvironment drives glioblastoma pathogenicity.

Malignant brain tumours are the cause of a disproportionate level of morbidity and mortality among cancer patients, an unfortunate statistic that has remained constant for decades. Despite considerable advances in the molecular characterization of these tumours, targeting the cancer cells has yet to produce significant advances in treatment. An alternative strategy is to target cells in the glioblastoma microenvironment, such as tumour-associated astrocytes. Astrocytes control multiple processes in health and disease, ranging from maintaining the brain's metabolic homeostasis, to modulating neuroinflammation. However, their role in glioblastoma pathogenicity is not well understood. Here we report that depletion of reactive astrocytes regresses glioblastoma and prolongs mouse survival. Analysis of the tumour-associated astrocyte translatome revealed astrocytes initiate transcriptional programmes that shape the immune and metabolic compartments in the glioma microenvironment. Specifically, their expression of CCL2 and CSF1 governs the recruitment of tumour-associated macrophages and promotes a pro-tumourigenic macrophage phenotype. Concomitantly, we demonstrate that astrocyte-derived cholesterol is key to glioma cell survival, and that targeting astrocytic cholesterol efflux, via ABCA1, halts tumour progression. In summary, astrocytes control glioblastoma pathogenicity by reprogramming the immunological properties of the tumour microenvironment and supporting the non-oncogenic metabolic dependency of glioblastoma on cholesterol. These findings suggest that targeting astrocyte immunometabolic signalling may be useful in treating this uniformly lethal brain tumour.

Alpha2beta1 Integrin Polymorphism in Diffuse Astrocytoma Patients.

Integrins are heterodimeric transmembrane glycoproteins resulting from the non-covalent association of an α and β chain. The major integrin receptor for collagen/laminin, α2β1 is expressed on a wide variety of cell types and plays an essential role in the adhesion of normal and tumor cells to the extracellular matrix. Integrin-triggered signaling pathways promote the invasion and survival of glioma cells by modifying the brain microenvironment. In this study, we investigated the association of a specific genetic polymorphism of integrin α2β1 with the incidence of diffusely infiltrating astrocytoma and the progression of these tumors. Single-nucleotide polymorphism in intron 7 of the integrin ITGA2 gene was examined in 158 patients and 162 controls using polymerase chain reaction and restriction enzyme analysis. The ITGA2 genotype +/+ (with a BglII restriction site in both alleles) exhibited higher frequency in grade II astrocytoma compared to control (P = 0.02) whereas the genotype -/- (lacking the BglII site) correlated with the poorest survival rate (P = 0.04). In addition, in silico analyses of ITGA2 expression from low-grade gliomas (LGG, n = 515) and glioblastomas (GBM, n = 159) indicated that the higher expression of ITGA2 in LGG was associated with poor overall survival (P < 0.0001). However, the distribution of integrin ITGA2 BglII genotypes (+/+, +/-, -/-) was not significantly different between astrocytoma subgroups III and IV (P = 0.65, 0.24 and 0.33; 0.29, 0.48, 0.25, respectively) compared to control. These results suggest a narrow association between the presence of this SNP and indicate that further studies with larger samples are warranted to analyze the relation between tumor grade and overall survival, highlighting the importance of determining these polymorphisms for prognosis of astrocytomas.

Effects of the m6Am methyltransferase PCIF1 on cell proliferation and survival in gliomas

BackgroundPrevious studies have suggested an important role for N6-methyladenosine (m6A) modification in the proliferation of glioma cells. N6, 2′-O-dimethyladenosine (m6Am) is another methylated form affecting the fate and function of most RNA. PCIF1 has recently been identified as the sole m6Am methyltransferase in mammalian mRNA. However, it remains unknown about the role of PCIF1 in the growth and survival of glioma cells. MethodsWe constructed glioma cell lines that stably downregulated/upregulated PCIF1, established intracranial xenograft models using these cell lines, and employed the following methods for investigations: CCK-8, EdU, colony formation, flow cytometry, qRT-PCR, Western blot, and immunohistochemistry. FindingsDownregulating PCIF1 promoted glioma cell proliferation, while overexpressing PCIF1 showed the opposite effects. Overexpression of PCIF1 blocked cell cycle progression and induced apoptosis in glioma cells, which was further confirmed by alterations in the expression of cell checkpoint proteins and apoptotic markers. Interestingly, disruption of PCIF1 methyltransferase activity slightly reversed the effect of PCIF1 overexpression on cell proliferation, but had no significant reversal effects on cell cycle progression or apoptosis. Knockdown of PCIF1 promoted the growth of gliomas, while overexpressing PCIF1 inhibited tumor growth and prolonged the survival time of tumor-bearing mice. In addition, the mRNA and protein levels of PCIF1 were gradually decreased with the increase of WHO grade in glioma tissues, but there was no significant correlation with patient survival. InterpretationThese results indicated that PCIF1 played a suppressing role in glioma growth and survival, which may not entirely depend on its methyltransferase activity.

Hispidulin Enhances Temozolomide (TMZ)-Induced Cytotoxicity against Malignant Glioma Cells In Vitro by Inhibiting Autophagy.

Temozolomide (TMZ), an oral alkylating agent, is the widely used first-line chemotherapeutic reagent for glioma in clinical practice. However, TMZ-induced autophagy is another cellular process favoring glioma cell survival. This study aimed to explore whether hispidulin can facilitate TMZ-induced cell death of glioma. The MTT assay showed that coadministration with hispidulin and TMZ could significantly decrease the viability of glioma U87MG cells. Meanwhile, hispidulin administration was also observed to promote TMZ-induced apoptosis. Furthermore, additional hispidulin treatment further elevated TMZ-induced expression of Bax, cleaved-caspase-9, and cleaved-caspase-3 protein but decreased Bcl-2 protein expression in U87MG cells. We also observed that hispidulin suppressed TMZ-induced autophagy to promote apoptosis, as showed by decreased AVOs and LC3B-I/II protein expression. These results collectively suggested that the combination of hispidulin and TMZ could improve the antitumor efficiency of TMZ against malignant gliomas.

Abstract 1867: Preclinical characterization of OSU- ERb-12, a novel ERβ agonist in Glioblastoma

Abstract Background: Glioblastoma (GBM) is the most aggressive primary brain tumor, representing approximately 15% of all primary CNS malignancies. Historically, the occurrence of GBM is higher in male than in female of reproductive age. Usage of exogenous hormones are known to reduce the risk of glioma development and estrogen improves survival in a glioblastoma animal models, suggesting a potential protective role in GBM. Estrogen receptor β (ERβ) may play a role as a tumor suppressor in GBM, and interestingly its expression decreases in higher-grade tumor samples with loss of differentiation. Therefore, a selective ERβ agonist could be a potential therapy against GBM. OSU-ERb-12 is brain penetrant, orally bioavailable ERβ agonist and demonstrates selectivity of ERβ: ERα = 46:1. In this work, we have tested the efficacy of this drug in vitro against glioma cells and in an orthotopic, syngeneic, immune-intact GBM mouse model. Materials &amp; Methods: We tested the efficacy of OSU-ERb-12, a novel selective ERβ agonist against commercial glioma cell lines and patient derived glioma stem like cells, cell proliferation rate via cell titer Glo assay. To evaluate the cell death Annexin-PI assay was performed using flow cytometry. We also tested impact of this drug on cell cycle distribution using flow cytometry-based assay. For in vivo studies, C57BL/6 mice were intracranially implanted with the murine glioma cell line GL261-Luc2 and treated with 30 mg/kg and 100 mg/kg of OSU-ERb-12. Tumor growth was evaluated post implantation using bioluminescent IVIS imaging and animal survival (Kaplan-Meier) was recorded. Results: Treatment with OSU-ERb-12 significantly reduced the cell proliferation rate of commercial cells and patient derived glioma stem cells from various molecular classifications with IC50= 5 µM, 72h. Cells treated with OSU-ERb-12 showed a time and dose dependent enhancement in programmed cell death. Activation of ERβ did not lead to any significant change in cell cycle pattern. Although treatment with 30 mg/kg OSU-ERb-12 did not show any change in survival (median survival 28 days) of tumor bearing mice, a significantly improved median survival was observed for mice dosed at 100 mg/kg (median survival 45 days) in comparison to vehicle (median survival 28 days). Conclusions: Our study demonstrates an important role of ERβ for glioma cell survival as observed by the potent biological effects of OSU-ERb-12 against glioma cell lines and immune intact murine glioma model that supports the potential for targeting the ERβ as a novel therapeutic strategy for treatment of GBM. Citation Format: Pratibha Sharma, Jayeeta Ghose, Christopher Coss, Chad Bennett, Raju R. Raval, Vinay Puduvalli. Preclinical characterization of OSU- ERb-12, a novel ERβ agonist in 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 1867.

PIK3CA Mutations in Diffuse Gliomas: An Update on Molecular Stratification, Prognosis, Recurrence, and Aggressiveness.

Introduction:PIK3CA is one of the most mutated oncogenes in solid tumors. In breast cancer (ER-positive, HER2-negative), these events represent a predictive biomarker of response to alpelisib. In glioblastomas (GBM), PIK3CA mutations were described as early constitutive events. Here, we investigated PIK3CA mutational profile across glioma molecular subgroups and its relevance during glioma recurrence. Furthermore, PIK3CA mutations’ effect in PI3K pathway, prognosis, and response to therapy was also explored.Material and Methods:Exons 10 and 21 of PIK3CA mutations were evaluated in 394 gliomas and 19 glioma recurrences from Instituto Português de Oncologia Lisboa Francisco Gentil (IPOLFG) and compared with The Cancer Genome Atlas (TCGA) data. TIMER2.0 and NetMHCpan4.1 were used to assess the immune-microenvironment contribution.Results:PIK3CA mutations were identified among all glioma subgroups, although with no impact on their stratification or prognosis. In both cohorts (IPOLFG and TCGA), PIK3CA mutation frequencies in IDH-mutant and IDH-wild-type GBM were similar (IPOLFG: 9% and 3%; TCGA: 8% and 2%). These mutations were not mutually exclusive with PTEN deletion and EGFR amplification. Despite their reduced frequency, we discovered PIK3CA mutations were maintained during glioma recurrence regardless of administered therapies. The immune microenvironment might not contribute to this phenotype as PIK3CA mutations did not influence immune cell infiltration.Conclusions:Despite the absence of a predominant effect in glioma stratification, PIK3CA mutations were maintained during glioma recurrence, possibly contributing to glioma cell survival, representing promising therapeutic targets in recurrent glioma. Nevertheless, understanding the potential synergistic effects between PIK3CA mutations, PTEN deletion, and EGFR amplification is pivotal to targeted therapies’ efficiency.

Ivermectin accelerates autophagic death of glioma cells by inhibiting glycolysis through blocking GLUT4 mediated JAK/STAT signaling pathway activation

This study aimed to investigate the regulatory effect of ivermectin (IVM) on energy metabolism in glioma progression, and provide a reference for the treatment of glioma. Glioma cells were treated with IVM to measure cell viability, autophagy marker protein expression, ATP content, glucose uptake, pyruvate content, and expression of key enzymes of glycolysis. Glucose transporter 4 (GLUT4) or siGLUT4 was transfected in IVM treated U87 cells to investigate the effect of GLUT4 on cellular glycolysis and autophagy. The JAK2 inhibitor AZD-1480 was introduced to explore the specific mechanism by which IVM regulates glycolysis and autophagy. Rat models of glioma xenograft were constructed and treated with 10mg/kg IVM to observe tumor growth and examine the expression levels of GLUT4 and autophagy marker proteins in tumor tissues. IVM inhibited glioma cell survival and promoted cell death. IVM promoted LC3-II protein expression and inhibited p62/SQSTM1 protein expression in glioma cells. IVM decreased adenosine-triphosphate (ATP) and pyruvate content, promoted glucose uptake, and reduced HK2 and PFK1 protein expression in U87 cells. IVM inhibited GLUT4 protein expression, and overexpression of GLUT4 promoted glycolysis and inhibited autophagic cell death in U87 cells. IVM inhibited glycolysis by blocking GLUT4 mediated the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway activation. IVM inhibited tumor growth in vivo, decreased the protein expression of GLUT4, JAK2, HK2, and PFK1 in tumor tissues, decreased the phosphorylation levels of STAT3/STAT5, and promoted the expression of autophagy marker proteins. IVM accelerates autophagic death of glioma cells by inhibiting glycolysis through blocking GLUT4 mediated JAK/STAT signaling pathway activation.

CBMS-5 One-carbon metabolism protect glioma cells under glutamine starvation through upregulation of MTHFD2

Cancer cells optimize nutrient utilization to supply energetic and biosynthetic pathways. However, less is known about how cancer cells exhibit metabolic flexibility to sustain cell growth and survival from nutrient starvation. Here, we find that serine and glycine levels were higher in low-nutrient regions of tumors in glioblastoma multiforme (GBM) patients than they were in other regions. Metabolic and functional studies demonstrated that serine availability and one-carbon metabolism support glioma cell survival following glutamine deprivation. Serine synthesis was mediated through autophagy rather than glycolysis. Gene expression analysis identified upregulation of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) to regulate one-carbon metabolism. In clinical samples, MTHFD2 expression was highest in the nutrient-poor areas around pseudopalisading necrosis. Genetic suppression of MTHFD2 and autophagy inhibition caused tumor cell death and growth inhibition of glioma cells upon glutamine deprivation. These results suggest new therapeutic targets for glioma cells adapting to a low-nutrient microenvironment.

TAMI-25. UPREGULATION OF CREATINE METABOLISM BY MYELOID CELLS RESULTS IN GLIOBLASTOMA PROGRESSION

Abstract Malignant brain tumors are uniquely immunosuppressive, with a predominant infiltration of immunosuppressive tumor-associated myeloid cells (TAMCs) and a deficit in T-cells unrivaled to any other tumor. This unique tumor microenvironment (TME) promotes resistance to both conventional and immune therapies for this disease. The underlying mechanisms by which TAMCs promote glioblastoma (GBM) progression are not fully understood. We found that TAMCs specifically upregulate de-novo creatine metabolism within GBM using unbiased genetic and metabolic screening. This metabolic phenotype was confirmed in human GBM patients by comparing peripheral versus tumor-infiltrating myeloid cells. Examination of de-novo creatine generation using Carbon13 arginine flux revealed that TAMCs, but not tumor-infiltrating CD8+ T-cells, can produce creatine. Furthermore, we demonstrate that TAMCs actively secrete de-novo generated creatine into cell cultures. Examination of the single-cell microenvironment of GBM revealed that malignant cells preferentially express the creatine transporter, indicating that TAMC-derived creatine is taken up by GBM. Notably, SLC6A8 is directly upregulated in the context of hypoxia and suggests that creatine uptake is a mechanism to promote survival under hypoxic stress. Indeed, exogenous creatine supplementation promoted both the migration and survival of multiple glioblastoma cell lines in-vitro. Utilizing an established inhibitor of creatine metabolism, β-Guanidinopropionic acid (β -GPA), we found that β -GPA blocks both the migration and survival of glioma cells under hypoxic stress. Lastly, β -GPA also inhibited creatine secretion by TAMCs, showing that creatine blockade can also influence TAMC metabolic phenotype. In the future, we will examine the importance of creatine metabolism on both immune suppression and tumor progression in-vivo. This work provides novel insights into the role of creatine metabolism in GBM and identifies a unique therapeutic avenue for this devastating disease.

TAMI-20. GLIOMA CELLS REPROGRAM SERINE-DEPENDENT ONE-CARBON METABOLISM TO SURVIVE GLUTAMINE STARVATION

Abstract Cancer cells optimize nutrient utilization to supply energetic and biosynthetic pathways. This metabolic process also includes redox maintenance and epigenetic regulation through nucleic acid and protein methylation, which enhance tumorigenicity and clinical resistance. However, less is known about how cancer cells exhibit metabolic flexibility to sustain cell growth and survival from nutrient starvation. Here, we find that serine and glycine levels were higher in low-nutrient regions of tumors in glioblastoma multiforme (GBM) patients than they were in other regions. Metabolic and functional studies in GBM cells demonstrated that serine availability and one-carbon metabolism support glioma cell survival following glutamine deprivation. Serine synthesis was mediated through autophagy rather than glycolysis. Gene expression analysis identified upregulation of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) to regulate one-carbon metabolism in GBM patient-derived sphere cells as well as in GBM cells. In clinical samples, MTHFD2 expression was highest in the nutrient-poor areas around “pseudopalisading necrosis.” Genetic suppression of MTHFD2 and autophagy inhibition caused tumor cell death and growth inhibition of glioma cells upon glutamine deprivation. These results may have important implications for serine-dependent one-carbon metabolism for glioma cells to survive glutamine starvation and suggest a new therapeutic strategy for patients with malignant glioma.

HAX1 maintains the glioma progression in hypoxia through promoting mitochondrial fission.

HCLS1‐associated protein X‐1 (HAX1), an anti‐apoptotic molecular, overexpresses in glioma. However, the role of HAX1 in glioma cell surviving in hypoxic environment remains unclear. Western blotting, qRT‐PCR, Transwell assay, TUNEL assay, wounding healing assay, clone formation, tumour xenograft model and immunohistochemical staining were used to investigate the role of HAX1 in glioma. HAX1 regulated by HIF‐1α was increased in glioma cells cultured in hypoxia. Silencing of HAX1 could cause an increased apoptosis of glioma cells cultured in hypoxia. Silencing of HAX1 also decreased the proliferation, migration and invasion of glioma cells cultured in hypoxia. Increased mitochondrial fission could prevent glioma cells from the damage induced by HAX1 knockdown in hypoxia. Furthermore, HAX1 was found to regulate glioma cells through phosphorylated AKT/Drp signal pathway. In conclusion, our study suggested that HAX1 promoted survival of glioma cells in hypoxic environment via AKT/Drp signal pathway. Our study also provided a potential therapeutic target for glioma.

LncRNA RP1-86C11.7 exacerbates the glioma progression and oncogenicity by hsa-miR-144-3p/TFRC signaling.

Glioblastoma (GBM) remains the most common and malignant tumor of the human central nervous system. Increasing evidence has highlighted that tumor cells with high transferrin receptor (TFRC) expression show advantages in growth. Long noncoding RNAs (lncRNAs) are related to glioma progression by mediating microRNAs (miRNAs). However, the underlying mechanism among TFRC, miRNA and lncRNA in GBM is limited. In the current study, we identified a new lncRNA-induced signaling mechanism that regulates the TFRC levels in GBM. The TFRC level was higher in glioma cell lines, and elevated TFRC expression promoted the proliferation and survival of glioma cells. Further study showed that hsa-miR-144a-3p bound to the 3′-UTR of TFRC mRNA and inhibited its expression, preventing the malignant properties of glioma cells, such as proliferation and survival. We also found that the lncRNA RP1-86C11.7 sponges hsa-miR-144-3p to suppress its protective role in glioma. RP1-86C11.7 overexpression in glioma cells elevated TFRC expression, increased the intracellular free iron level, and deteriorated oncogenicity, with a significant reduction in hsa-miR-144-3p. By contrast, silencing RP1-86C11.7 upregulated the hsa-miR-144-3p level, resulting in decreased TFRC expression and repressed glioma progression. However, the effect of silencing RP1-86C11.7 was reversed with simultaneous hsa-miR-144-3p inhibitor treatment: the TFRC level, intracellular iron level and proliferation in glioma cells increased. Mechanistically, our data indicated that RP1-86C11.7 exacerbates the malignant behavior of glioma through the hsa-miR-144-3p/TFRC axis. RP1-86C11.7 may be a potential biomarker or target to treat glioma in the future.

CircVCAN/SUB1 up-regulates MYC/HSP90β to enhance the proliferation and migration of glioma cells

BackgroundGliomas are the most malignant and aggressive form of brain tumors, and account for the majority of brain cancer related deaths. Previous studies have reported that SUB1 serves as a novel RBP, which is highly expressed in tumors. AimIn our research, we aimed at exploring the regulatory mechanism of SUB1 in glioma cells. MethodsRT-qPCR was conducted to measure the expression of SUB1, HSP90β and MYC in glioma cells in U87 cell. Cell function tests were conducted to identify how the ectopic SUB1 expression influenced the proliferation and migration of glioma cells. Also, functional assays and mechanism experiments were carried out to figure out the relationship among circVCAN, SUB1 and MYC/HSP90β. ResultsSUB1 expression was up-regulated in glioma cells. The inhibition of SUB1 expression suppressed the proliferation and migration of glioma cells, while the up-regulation of that accelerated the proliferation and migration of glioma cells. CircVCAN could up-regulate the expression of MYC/HSP90β by recruiting SUB1, thereby promoting glioma progression. ConclusionSUB1 plays a significant role in the survival and migration of glioma cells.