Differentiation Of Glioma Cells Research Articles

Endoplasmic Reticulum Stress Contributed to Dipyridamole-Induced Impaired Autophagic Flux and Glioma Apoptosis.

Elevation of intracellular cAMP levels has been implicated in glioma cell proliferation inhibition, differentiation, and apoptosis. Inhibition of phosphodiesterase is a way to elevate intracellular cAMP levels. The present study aimed to investigate the anti-glioma potential of dipyridamole, an inhibitor of phosphodiesterase. Upon treatment with dipyridamole, human U87 glioma cells decreased cell viability, clonogenic colonization, migration, and invasion, along with Noxa upregulation, Endoplasmic Reticulum (ER) stress, impaired autophagic flux, Yes-associated Protein 1 (YAP1) phosphorylation, and YAP1 reduction. Pharmacological and genetic studies revealed the ability of dipyridamole to initiate Noxa-guided apoptosis through ER stress. Additionally, the current study further identified the biochemical role of YAP1 in communicating with ER stress and autophagy under situations of dipyridamole treatment. YAP1 promoted autophagy and protected glioma cells from dipyridamole-induced apoptotic cell death. Dipyridamole impaired autophagic flux and rendered glioma cells more vulnerable to apoptotic cell death through ER stress-inhibitable YAP1/autophagy axis. The overall cellular changes caused by dipyridamole appeared to ensure a successful completion of apoptosis. Dipyridamole also duplicated the biochemical changes and apoptosis in glioma T98G cells. Since dipyridamole has additional biochemical and pharmacological properties, further research centered on the anti-glioma mechanisms of dipyridamole is still needed.

Organophosphates modulate tissue transglutaminase activity in differentiated C6 neural cells.

The organophosphate compounds chlorpyrifos (O, O-diethyl O-[3,5,6-trichloro-2-pyridinyl] phosphorothioate, CPF) and phenyl saligenin phosphate (PSP) have been widely implicated in developmental neurotoxicity and neurodegeneration. However, the underlying mechanism remains unclear. Transglutaminase (TG)2 is a calcium ion (Ca2+)-dependent enzyme with an important role in neuronal cell outgrowth and differentiation and in neurotoxin activity and is modulated by organophosphates. We studied TG2 activity modulation by CPO and PSP during differentiation in C6 glioma cells. We studied the effects of CPO or PSP treatment with or without the TG2 inhibitor Z-DON and identified potential TG2 protein substrates via mass spectrometry. PSP and CPO did not affect cell viability but affected TG2 activity in differentiating cells. Our results indicate that the organophosphate-induced amine incorporation activity of TG2 may have a direct effect on neuronal outgrowth, differentiation, and cell survival by modifying several essential microtubule proteins, including tubulin. Inhibiting TG2 reduced neurite length but not cell survival. TG2 inhibitors can protect against organophosphate-induced neuropathy and could be used for developing novel therapeutic strategies for treating brain cancer and neurodegenerative disorders.

HIF1α/HIF2α induces glioma cell dedifferentiation into cancer stem cells through Sox2 under hypoxic conditions.

Objective: Our previous study showed that glioma stem-like cells could be induced to undergo dedifferentiation under hypoxic conditions, but the mechanism requires further study. HIF1α and HIF2α are the main molecules involved in the response to hypoxia, and Sox2, as a retroelement, plays an important role in the formation of induced pluripotent stem cells, especially in hypoxic microenvironments. Therefore, we performed a series of experiments to verify whether HIF1α, HIF2α and Sox2 regulated glioma cell dedifferentiation under hypoxic conditions.Materials and methods: Sphere formation by single glioma cells was observed, and CD133 and CD15 expression was compared between the normoxic and hypoxic groups. HIF1α, HIF2α, and Sox2 expression was detected using the CGGA database, and the correlation among HIF1α, HIF2α and Sox2 levels was analyzed. We knocked out HIF1α, HIF2α and Sox2 in glioma cells and cultured them under hypoxic conditions to detect CD133 and CD15 expression. The above cells were implanted into mouse brains to analyze tumor volume and survival time.Results: New spheres were formed from single glioma cells in 1% O2, but no spheres were formed in 21% O2. The cells cultured in 1% O2 highly expressed CD133 and CD15 and had a lower apoptosis rate. The CGGA database showed HIF1α and HIF2α expression in glioma. Knocking out HIF1α or HIF2α led to a decrease in CD133 and CD15 expression and inhibited sphere formation under hypoxic conditions. Moreover, tumor volume and weight decreased after HIF1α or HIF2α knockout with the same temozolomide treatment. Sox2 was also highly expressed in glioma, and there was a positive correlation between the HIF1α/HIF2α and Sox2 expression levels. Sox2 was expressed at lower levels after HIF1α or HIF2α was knocked out. Then, Sox2 was knocked out, and we found that CD133 and CD15 expression was decreased. Moreover, a lower sphere formation rate, higher apoptosis rate, lower tumor formation rate and longer survival time after temozolomide treatment were detected in the Sox2 knockout cells.Conclusion: In a hypoxic microenvironment, the HIF1α/HIF2α-Sox2 network induced the formation of glioma stem cells through the dedifferentiation of differentiated glioma cells, thus promoting glioma cell chemoresistance. This study demonstrates that both HIF1α and HIF2α, as genes upstream of Sox2, regulate the malignant progression of glioma through dedifferentiation.

Human Glioma Cells Therapy Using ATRA-Induced Differentiation Method to Promote the Inhibitive Effect of TMZ and CCDP.

The glioma stem cells (GSCs) performed the self-renewal, proliferation, and differentiation characteristics; their drug resistance has become the main reason for glioma clinical treatment failure. All-trans retinoic acid (ATRA) is an important inducer of cell differentiation, applied in the treatment of hematologic diseases and other solid tumors. ATRA is a fat-soluble compound, which can easily go through the blood-brain barrier. Therefore, in this study, ATRA was used to induce the differentiation of glioma cells and glioma stem cells, reducing the degree of malignancy and improving its chemotherapy resistance. Methods and Treatment. The results of IF and PCR showed that the expression of CD133 was significantly lower than those of undifferentiated cells. Furthermore, temozolomide (TMZ) and cisplatin (CDDP), the first-line drugs, were used for the treatment of GCs and GSCs. The MTT assay results showed that the effect of the combination of the two drugs was significantly stronger than that of one of them alone. Results. Moreover, the MTT assay also demonstrated that TMZ single, CDDP single, and the combination of TMZ and CDDP can inhibit the proliferation of GCs, ATRA-GCs, GSCs, and ATRA-GSCs in a dose- and time-dependent manner; and ATRA-induced differentiation could promote those drugs inhibition effect and increased the chemotherapy sensitivity. Conclusion. Therefore, we successfully purified the suspension spherical glioma stem cells. Moreover, ATRA was demonstrated to induce the differentiation of GCs and GSCs. Furthermore, ATRA-induced differentiation promotes the inhibitive effect of TMZ and CCDP treatment on the proliferation of primary human glioma cells and glioma stem cells, suggesting that ATRA could increase the chemotherapy sensitivity of TMZ and CCDP through inducing cell differentiation. The combination of TMZ and CCDP performed a synergistic role in inhibiting the proliferation of GCs and GSCs.

MiR-221/222 suppression induced by activation of the cAMP/PKA/CREB1 pathway is required for cAMP-induced bidirectional differentiation of glioma cells.

Factors that increase cAMP levels can induce lineage-specific differentiation of glioma cells into astrocyte-like cells. However, the differentiation pattern and underlying mechanisms remain unclear. Here, we find that cAMP/protein kinase A (PKA)/cAMP responsive element binding protein 1 (CREB1)-induced miR-221/222 suppression contributes to the neuron-like differentiation of gliomas. cAMP agonists selectively induced neuron- and astrocyte-like but not oligodendrocyte-like differentiation of C6 glioma cells. PKA inhibitors and CREB1 knockout blocked neuron-like differentiation of glioma cells. cAMP inhibited miR-221/222 in a PKA/CREB1-dependent manner. Importantly, both in vitro and in vivo assays demonstrated that transcriptional suppression of miR-221/222 is required for neuronal differentiation of glioma cells. Our findings suggest that increasing cAMP levels can induce bidirectional differentiation of glioma cells. Furthermore, the miR-221/222 cluster acts as an epigenetic brake during glioma differentiation.

In silico identification of natural product inhibitors against Octamer-binding transcription factor 4 (Oct4) to impede the mechanism of glioma stem cells.

Octamer-binding transcription factor 4 (Oct4) is a core regulator in the retention of stemness, invasive, and self-renewal properties in glioma initiating cells (GSCs) and its overexpression inhibits the differentiation of glioma cells promoting tumor cell proliferation. The Pit-Oct-Unc (POU) domain comprising POU-specific domain (POUS) and POU-type homeodomain (POUHD) subdomains is the most critical part of the Oct4 for the generation of induced pluripotent stem cells from somatic cells that lead to tumor initiation, invasion, posttreatment relapse, and therapeutic resistance. Therefore, the present investigation hunts for natural product inhibitors (NPIs) against the POUHD domain of Oct4 by employing receptor-based virtual screening (RBVS) followed by binding free energy calculation and molecular dynamics simulation (MDS). RBVS provided 13 compounds with acceptable ranges of pharmacokinetic properties and good docking scores having key interactions with the POUHD domain. More Specifically, conformational and interaction stability analysis of 13 compounds through MDS unveiled two compounds ZINC02145000 and ZINC32124203 which stabilized the backbone of protein even in the presence of linker and POUS domain. Additionally, ZINC02145000 and ZINC32124203 exhibited stable and strong interactions with key residues W277, R242, and R234 of the POUHD domain even in dynamic conditions. Interestingly, ZINC02145000 and ZINC32124203 established communication not only with the POUHD domain but also with the POUS domain indicating their incredible potency toward thwarting the function of Oct4. ZINC02145000 and ZINC32124203 also reduced the flexibility and escalated the correlations between the amino acid residues of Oct4 evidenced by PCA and DCCM analysis. Finally, our examination proposed two NPIs that can impede the Oct4 function and may help to improve overall survival, diminish tumor relapse, and achieve a cure not only in deadly disease GBM but also in other cancers with minimal side effects.

PAK3 is a key signature gene of the glioma proneural subtype and affects its proliferation, differentiation and growth.

Gliomas are the most lethal adult primary brain cancers. Recent advances in their molecular characterization have contributed to a better understanding of their pathophysiology, but there is still a need to identify key genes controling glioma cell proliferation and differentiation. The p21-activated kinases PAK1 and PAK2 play essential roles in cell division and brain development and are well-known oncogenes. In contrast, the role of PAK3 in cancer is poorly understood. It is known, however, that this gene is involved in brain ontogenesis and has been identified as a gene of the proneural subtype signature in glioblastomas. To better understand the role of PAK kinases in the pathophysiology of gliomas, we conducted expression analyses by querying multiple gene expression databases and analyzing primary human glioma samples. We next studied PAK3 expression upon differentiation in patient-derived cell lines (PDCLs) and the effects of PAK3 inhibition by lentiviral-mediated shRNA on glioma cell proliferation, differentiation and tumor growth. We show that contrary to PAK1 and PAK2, high PAK3 expression positively correlates with a longer survival of glioma patients. We also found that PAK3 displays differential expression patterns between glioma sub-groups with a higher expression in 1p/19q-codeleted oligodendrogliomas, and is highly expressed in tumors and PDCLs of the proneural subtype. In PDCLs, high PAK3 expression negatively correlated with proliferation and positively correlated with neuronal differentiation. Inhibition of PAK3 expression increased PDCL proliferation and glioma tumor growth in nude mice. Our results indicate that PAK3 plays a unique role among PAKs in glioma development and may represent a potential therapeutic target.

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

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

Differential expression of stem cell markers in proliferating cells in glioma

PurposeThe identification of prognostically and therapeutically relevant molecular markers is fundamental to the further development of personalised therapies in brain tumours. Current therapeutic options for the treatment of gliomas rely mainly on surgical resection and the inhibition of tumour cell proliferation by irradiation and chemotherapy. Glioma stem cells are a subpopulation of proliferating tumour cells that have self-renewal capacity and can give rise to heterogeneous cells that comprise the tumour and are thought to play a role in the resistance of gliomas to therapy. The aim of this study was to evaluate the expression of markers of glioma stem cells and differentiated glial cells in proliferating glioma cells in comparison to the overall expression of the respective markers in the tumour tissue.MethodsTissue microarrays were assembled from specimen of pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, glioblastoma, oligodendroglioma, anaplastic oligodendroglioma, ependymoma, and anaplastic ependymoma. These were immunohistochemically double stained with antibodies against the proliferation-associated antigen Ki67 and marker proteins for glioma stem cells (CD133, Nestin, Musashi, CD15, CD44), and differentiated glioma cells (GFAP, MAP2c).ResultsThe expression of both glial and glioma stem cell markers differs between proliferating and non-proliferating glioma cells. Furthermore, the proliferating cells in the different glial tumour entities show a different expression profile.ConclusionFurther analysis of marker expression in proliferating glioma cells and correlation with clinical outcome and susceptibility to irradiation and chemotherapy might help establish new biomarkers and therapies for glioma.

HDAC6 Signaling at Primary Cilia Promotes Proliferation and Restricts Differentiation of Glioma Cells.

Simple SummaryGlioblastoma is the most common and lethal brain tumor in adults because it becomes resistant to virtually every treatment. Histone deacetylase 6 (HDAC6), which is located primarily in the cytoplasm, has a unique role in promoting the disassembly of cells’ primary cilium, a non-motile “antenna” that must be broken down before cells can progress through the cell cycle. The role of HDAC6 and its function in gliomas have not been investigated with respect to tumor cell cilia. We have found that inhibitors of HDAC6 cause rapid and specific changes inside glioma cilia, reducing tumor cell proliferative capacity and promoting cell differentiation. Importantly, the HDAC6 inhibitors did not affect the proliferation or differentiation of glioma cells that we genetically modified unable to grow cilia. Our findings reveal a conserved and critical role for HDAC6 in glioma growth that is dependent on cilia.Histone deacetylase 6 (HDAC6) is an emerging therapeutic target that is overexpressed in glioblastoma when compared to other HDACs. HDAC6 catalyzes the deacetylation of alpha-tubulin and mediates the disassembly of primary cilia, a process required for cell cycle progression. HDAC6 inhibition disrupts glioma proliferation, but whether this effect is dependent on tumor cell primary cilia is unknown. We found that HDAC6 inhibitors ACY-1215 (1215) and ACY-738 (738) inhibited the proliferation of multiple patient-derived and mouse glioma cells. While both inhibitors triggered rapid increases in acetylated alpha-tubulin (aaTub) in the cytosol and led to increased frequencies of primary cilia, they unexpectedly reduced the levels of aaTub in the cilia. To test whether the antiproliferative effects of HDAC6 inhibitors are dependent on tumor cell cilia, we generated patient-derived glioma lines devoid of cilia through depletion of ciliogenesis genes ARL13B or KIF3A. At low concentrations, 1215 or 738 did not decrease the proliferation of cilia-depleted cells. Moreover, the differentiation of glioma cells that was induced by HDAC6 inhibition did not occur after the inhibition of cilia formation. These data suggest HDAC6 signaling at primary cilia promotes the proliferation of glioma cells by restricting their ability to differentiate. Surprisingly, overexpressing HDAC6 did not reduce cilia length or the frequency of ciliated glioma cells, suggesting other factors are required to control HDAC6-mediated cilia disassembly in glioma cells. Collectively, our findings suggest that HDAC6 promotes the proliferation of glioma cells through primary cilia.

Differentiated Cancer Cell-Derived Fibromodulin Promotes Angiogenesis and Tumor Growth Through Paracrine Signaling in Endothelial Cells

Cancer stem-like cells alone can initiate tumors, but the function of differentiated cancer cells that form the tumor bulk remains poorly understood. Proteomic analysis showed a higher abundance of the collagen-binding proteoglycan Fibromodulin (FMOD) in the conditioned medium of differentiated glioma cells (DGCs) compared to that of glioma stem-like cells (GSCs). DGCs silenced for FMOD fail to cooperate with co-implanted GSCs to promote tumor growth. FMOD downregulation neither affects GSC growth and differentiation nor DGC growth and reprogramming in vitro. DGC-secreted FMOD promotes angiogenesis, which requires activation of Integrin-dependent Notch signaling in endothelial cells. Furthermore, conditional silencing of FMOD in newly generated DGCs in vivo inhibits the growth of GSC-initiated tumors due to poorly developed vasculature and increases mouse survival. Collectively, these findings demonstrate that DGC-secreted FMOD promotes glioma tumor angiogenesis and growth through paracrine signaling in endothelial cells and identifies a DGC-produced protein as a potential therapeutic target in glioma.

MiRNA-mediated loss of m6A increases nascent translation in glioblastoma.

Within the glioblastoma cellular niche, glioma stem cells (GSCs) can give rise to differentiated glioma cells (DGCs) and, when necessary, DGCs can reciprocally give rise to GSCs to maintain the cellular equilibrium necessary for optimal tumor growth. Here, using ribosome profiling, transcriptome and m6A RNA sequencing, we show that GSCs from patients with different subtypes of glioblastoma share a set of transcripts, which exhibit a pattern of m6A loss and increased protein translation during differentiation. The target sequences of a group of miRNAs overlap the canonical RRACH m6A motifs of these transcripts, many of which confer a survival advantage in glioblastoma. Ectopic expression of the RRACH-binding miR-145 induces loss of m6A, formation of FTO/AGO1/ILF3/miR-145 complexes on a clinically relevant tumor suppressor gene (CLIP3) and significant increase in its nascent translation. Inhibition of miR-145 maintains RRACH m6A levels of CLIP3 and inhibits its nascent translation. This study highlights a critical role of miRNAs in assembling complexes for m6A demethylation and induction of protein translation during GSC state transition.

Lidocaine inhibits glioma cell proliferation, migration and invasion by modulating the circEZH2/miR-181b-5p pathway.

Lidocaine is well known as a local anesthetic that has been reported to play an antitumor role in numerous cancers, including glioma. Circular RNAs (circRNAs) play multiple biological roles in cancers. The aim of this study was to determine the effects of lidocaine in glioma in vitro and in vivo and explore functional mechanisms. The effects of lidocaine on glioma progression were investigated by cell proliferation, migration and invasion using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, colony formation assay and transwell assay. The expression of CD133 and glial fibrillary acidic protein (GFAP) was quantified by western blot to assess cell differentiation. The expression of circEZH2 and miR-181b-5p was detected by a quantitative real-time PCR (qRT-PCR). The target relationship between circEZH2 and miR-181b-5p was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The effect of lidocaine on tumor growth in vivo was investigated by establishing Xenograft models. Lidocaine inhibited proliferation, migration, invasion and induced differentiation of glioma cells in vitro. Lidocaine suppressed the expression of circEZH2, and circEZH2 was highly expressed in glioma tissues and cells. CircEZH2 overexpression partly inhibited the function of lidocaine. CircEZH2 was a sponge of miR-181b-5p, and miR-181b-5p was downregulated in glioma tissues and cells. Besides, miR-181b-5p restoration reversed the effects of circEZH2 overexpression to repress the malignant behaviors of glioma cells. In addition, lidocaine mediated the circEZH2/miR-181b-5p axis to inhibit tumor growth in vivo. Lidocaine suppressed glioma progression by modulating the circEZH2/miR-181b-5p pathway.

Differentiation of Tumorigenic C6 Glioma Cells Induced by Enhanced IL-6 Signaling.

Background and objectives: Cancer stem cells (CSCs) are obstacles to cancer therapy due to their therapeutic resistance, ability to initiate neoplasia, and roles in tumor relapse and metastasis. Efforts have been made to cure CSCs, such as the use of differentiation therapy, which induces cancer stem-like cells to undergo differentiation and decrease their tumorigenicity. Interleukin 6 (IL-6) upregulates the expression of glial fibrillary acidic protein (GFAP) in C6 glioma cells, indicating that it is able to induce the differentiation of these cells. The C6 glioma cell line forms a high percentage of cancer stem-like cells, leading us to speculate whether IL-6 signaling could modulate the differentiation of tumorigenic C6 glioma cells. However, we observed that IL-6 alone could not efficiently induce the differentiation of these cells. Therefore, different IL-6 signaling elicitors, including IL-6 alone, a combination of IL-6 and soluble IL-6 receptor (IL-6/sIL-6R), and tumor necrosis factor-α (TNF-α) plus IL-6/sIL-6R (TNF-α/IL-6/sIL-6R), were evaluated for their potential use in differentiation therapy. Materials and Methods: The potential of IL-6 signaling elicitors in differentiation therapy were examined by assessing changes in biomarker levels, the rate of cell proliferation, and tumorigenicity, respectively. Results: Enhanced IL-6 signaling could effectively induce C6 glioma cell differentiation, as determined by observed variations in the expression of differentiation, cell cycle, and stem cell biomarkers. Additionally, the total cell population and the tumorigenicity of glioma cells were all considerably reduced after TNF-α/IL-6/sIL-6R treatment. Conclusions: Our findings provide evidence that enhanced IL-6 signaling can efficiently promote tumorigenic C6 glioma cells to undergo differentiation.

Knockdown of TXNDC9 induces apoptosis and autophagy in glioma and mediates cell differentiation by p53 activation.

Glioma is the most common malignant brain tumor. Because of its high degree of malignancy, the effect of surgical treatment, radiotherapy, chemotherapy, or immunotherapy is not ideal. TXNDC9 belongs to thioredoxin domain-containing proteins, which is involved in tumor progression. However, no research associated with TXNDC9 has been reported in glioma. In this study, we found that TXNDC9 was upregulated in glioma. Knockdown of TXNDC9 would prevent proliferation and metastasis, induce the apoptosis rate of glioma cells, and promote the expression Cleaved-caspase3, Cleaved-caspase8, Cleaved-caspase9. Meanwhile, knockdown of TXNDC9 induced autophagy by increasing the level of LC3 and Beclin-1. Cell morphology and expression analysis of GFAP, Vimentin, verified that TXNDC9 could regulate glioma cell differentiation. During this program, the expression of p53 changes dramatically. The apoptosis, autophagy, and cell differentiation program were blocked by p53 inhibitor treatment. In conclusion, the silencing of TXNDC9 induces apoptosis and autophagy in glioma and promotes cell differentiation by controlling p53 and may function as a new mechanism in glioma.

Abstract 6611: Functional characterization of a novel gene in the antigen presentation pathway allows specific immunotherapeutic targeting of glioblastoma cancer stem cells

Abstract Glioblastoma is the most prevalent and aggressive primary brain tumor in adults, and conventional treatment strategies have made minimal progress in prolonging overall survival. Cancer stem cells are critical for tumor initiation, growth, and maintenance, and are able to co-opt complex host mechanisms to evade immune recognition. Immune checkpoint inhibition has proven revolutionary for the treatment of several types of solid tumors, but has largely failed in attempts to improve outcomes in glioblastoma. Developing a deeper understanding of the functional immunomodulatory role of genes unique to glioma stem cells (GSCs) may help to elucidate this enigma, and inform more specific immunotherapeutic targeting. To better understand the ability of GSCs to promote immunosuppression and self-tolerance in the tumor niche, we interrogated RNA-seq data from GSCs and differentiated glioma cells (DGCs) and identified a novel gene in the antigen presentation pathway (APP). In vitro targeting of this gene increased sensitivity to CD8+ T cell killing and reduced expression of other T-cell inhibitory ligands in GSCs alone, decreasing tumor-mediated immunosuppression. Compared to DGCs, GSCs were found to be key promoters of immune self-tolerance and evasion, with unaltered GSCs exhibiting minimal peptide binding to MHC Class I and, therefore, decreased antigen presentation. Specific targeting of the identified APP gene led to a reversal of this interaction, with increased binding capacity and, correspondingly, tumor antigen recognition. In vivo administration of a specific APP gene targeting agent in human GSC xenograft mice displayed decreased tumor burden and a striking prolongation of overall survival compared to both controls and mice treated with known immune checkpoint inhibitors. High expression of the APP gene is associated with poor clinical prognosis, supporting the clinical relevance of this discovery. Thus, novel targeting of the antigen-presenting pathway may provide a therapeutic vulnerability unique to glioblastoma cancer stem cells. Citation Format: Reilly L. Kidwell, Xiqing Li, Jeremy N. Rich. Functional characterization of a novel gene in the antigen presentation pathway allows specific immunotherapeutic targeting of glioblastoma cancer stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6611.

Tailored Lipoprotein-Like miRNA Delivery Nanostructure Suppresses Glioma Stemness and Drug Resistance through Receptor-Stimulated Macropinocytosis.

Glioma initiating cells (GICs) function as the seed for the propagation and relapse of glioma. Designing a smart and efficient strategy to target the GICs and to suppress the multiple signaling pathways associated with stemness and chemoresistance is essential to achieving a cancer cure. Inspired by the metabolic difference in endocytosis between GICs, differentiated glioma cells, and normal cells, a tailored lipoprotein‐like nanostructure is developed to amplify their internalization into GICs through receptor‐stimulated macropinocytosis. As CXCR4 is highly expressed on GICs and glioma tumor sites, meanwhile, the activation of CXCR4 induces the receptor‐stimulated macropinocytosis pathway in GICs, this CXCR4 receptor‐stimulated lipoprotein‐like nanoparticle (SLNP) achieves efficient accumulation in GICs in vitro and in vivo. By carrying microRNA‐34a in the core, this tailored SLNP reduces sex‐determining region Y‐box 2 and Notch1 expression, powerfully inhibits GICs stemness and chemoresistance, and significantly prolongs the survival of GICs‐bearing mice. Taken together, a tailored lipoprotein‐based nanostructure realizes efficient GICs accumulation and therapeutic effect through receptor‐stimulated macropinocytosis, providing a powerful nanoplatform for RNA interference drugs to combat glioma.

A doxycycline-inducible C17.2 neural stem cell-based combination of differentiation and suicide gene therapy for an in vitro tumorigenic C6 glioma model

Neural stem cell (NSC)-based gene therapies have been recently developed as effective strategies for treating brain tumors through inherent tumor tropism. However, this methodology has two considerable challenges: preventing NSCs from dying from the therapeutic agents encoded by their equipped genes before reaching tumor sites and the clearance of exogenous NSCs after therapeutic treatments. For these purposes, we established a novel doxycycline-inducible retroviral plasmid pTRE3G-TKGFP. A herpes simplex type 1 thymidine kinase (HSV1TK)-green fluorescent protein (GFP) fusion protein coding sequence was integrated into the multiple cloning site II (MCS II) of the pQcXIX vector, and the CMV IE promoter (PCMV IE) of pQcXIX was replaced with the doxycycline-inducible TRE3G promoter (PTRE3G). We then cloned the coding sequences of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6) and soluble interleukin 6 receptor (sIL-6R) into MCS I to combine HSV1TK (TK)/ganciclovir (GCV)-based suicide gene therapy against tumorigenic C6 glioma cells. TNF-α, IL-6 and sIL6R could efficiently induce tumorigenic C6 glioma cell differentiation, resulted in down-regulating the cell proliferation rate and the tumorigenicity of glioma cells and up-regulating the production of differentiation markers, such as connexin-43. Furthermore, gap junctions could enhance the bystander effect in suicide gene therapy. Consequently, we found that the retroviral plasmid transfected NSCs exerted stronger remedial effects on tumorigenic C6 glioma cells through the combination of differentiation and suicide gene therapy than by suicide gene therapy alone. This study is also the first case applying NSCs to conduct the combination of differentiation and TK/GCV-based suicide gene therapy on glioma cells.

STEM-10. BIDIRECTIONAL INTERACTION BETWEEN TUMOR-ASSOCIATED PLATELETS AND GLIOMA STEM CELLS IN GLIOBLASTOMA MULTIFORME

Abstract Glioblastoma Multiforme (GBM) is the most common and lethal malignant primary adult brain tumor. Therapy resistance and tumor recurrence in GBM have been attributed to glioma stem cells (GSCs), which are found in hypoxic but perivascular niches. We hypothesized that the clinically documented and prognostically relevant increase in platelets in GBM promotes formation of functional hypoxic but perivascular niches that support GSCs due to formation of intravascular thromboses, a hallmark of GBM. Our preliminary in silico analysis from TCGA GBM samples indicates significant correlation of platelet and stemness signature expression (P < 0.001). High expression of platelet gene signatures also inversely correlates with overall survival in GBM patients (P < 0.02). Furthermore, we found significant co-localization of known platelet and stemness markers in primary GBM specimens, supporting our hypothesis and motivating further interrogation of GSC-platelet crosstalk. Our preliminary data suggest that GSCs exposed to either tumor or healthy platelets demonstrate increased self-renewal and stemness, as determined by a significant increase in OCT4, NANOG, and OLIG2 expression compared to unexposed GSCs. This increase in stemness and self-renewal markers was not seen in platelet-exposed differentiated glioma cell (DGC) counterparts. Conversely, platelets are stimulated by GSCs and GSC-conditioned media while DGCs elicit no stimulatory effects as measured by ATP release and aggregation assays. Our results implicate a functional role for platelet-GSC interactions in the maintenance of tumor cellular hierarchy that ultimately contributes to poor clinical outcomes in GBM. RNA-Seq of platelet-privileged GSCs is currently underway to elucidate the mechanism by which platelets impact GSC self-renewal. Successful characterization of potential crosstalk between platelets and GSCs may offer new clinical perspectives into GBM and inform development of a novel treatment paradigm to target these specific cell-to-cell interactions.

STEM-25. HDAC1 IS ESSENTIAL FOR GLIOMA STEM CELL SURVIVAL

Abstract OLIG2 is a central nervous system-specific transcription factor that is expressed in almost all diffuse gliomas. It is also one of the key core transcription factors that can reprogram differentiated glioma cells to highly tumorigenic glioma stem-like cells (GSCs). We have previously shown that expression of OLIG2 is critical for glioma growth both in a genetically relevant mouse model as well as in patient-derived xenograft models. Our work suggests that a small molecule inhibitor of OLIG2 could serve as a highly targeted therapy for high-grade glioma; however, transcription factors are generally very difficult to target because their interactions with DNA and co-regulatory proteins involve large and complex surface area contacts. Our laboratory has shown that OLIG2 functions are regulated through interactions with distinct co-regulator proteins in normal neural stem cells. However, there are currently no reports on interactors that promote the proto-oncogenic functions of OLIG2 in malignant glioma. In this study, we employed two independent proteomics screens identify tumor-specific, druggable OLIG2 co-regulators as possible surrogate targets to suppress OLIG2 function in glioma. These screens led to the identification of a novel OLIG2 partner protein: Histone Deacetylase 1 (HDAC1). We confirmed that this interaction occurs in both murine and human glioma models. Although HDACs are ubiquitously expressed and are known to be functionally redundant, we show that ablation of HDAC1 alone significantly decreases the stemness and proliferation capacity of patient-derived GSCs in a p53-dependent manner, while having a minimal impact on normal human neural stem cells and astrocytes. Furthermore, we demonstrate that knockdown of HDAC1, in combination with ionizing radiation treatment, significantly alters the growth pattern of intracranial tumors in vivo. We demonstrate that HDAC1 function is critical for GSC growth and provide a strong rationale for targeting the OLIG2-HDAC1 interaction in malignant glioma.