Glioma Stem-like Cells Research Articles

NDGA-P21, a novel derivative of nordihydroguaiaretic acid, inhibits glioma cell proliferation and stemness

Nordihydroguaiaretic acid (NDGA) and its synthetic chiral analog dl-nordihydroguaiaretic acid (Nordy) show collective benefits in anti-tumor, and defending against viral and bacterial infections. Here, we synthetized a new derivative-NDGA-P21 based on NDGA structure. Regardless of the structural similarity, NDGA-P21 exhibited stronger capability in suppression of glioblastoma (GBM) cell growth as compared to Nordy. Mechanically, NDGA-P21 is able to arrest cell cycle of GBM cells in G0/G1 phase, and to block cell proliferation sequentially. It is important to note that NDGA-P21 is able to impair the stemness of glioma stem-like cells (GSLCs) via measurement of colony formation and sphere formation. Taken together, the novel NDGA-based compound NDGA-P21 exhibits potential therty -20 apeutic implications through inhibiting proliferation of glioma cells and self-renewal capability of GSLCs.

EZH2 Palmitoylation Mediated by ZDHHC5 in p53-Mutant Glioma Drives Malignant Development and Progression.

Gliomas with mutant p53 occurring in 30% of glioma patients exhibit therapeutic resistance and poor outcomes. In this study, we identify a novel mechanism through which mutant p53 drives cancer cell survival and malignant growth. We documented overexpression of the zinc finger protein ZDHHC5 in glioma compared with normal brain tissue and that this event tightly correlated with p53 mutations. Mechanistic investigations revealed that mutant p53 transcriptionally upregulated ZDHHC5 along with the nuclear transcription factor NF-Y. These events contributed to the development of glioma by promoting the self-renewal capacity and tumorigenicity of glioma stem-like cells, by altering the palmitoylation and phosphorylation status of the tumor suppressor EZH2. Taken together, our work highlighted ZDHHC5 as a candidate therapeutic target for management of p53-mutated gliomas. Cancer Res; 77(18); 4998-5010. ©2017 AACR.

Chronic D609 treatment interferes with cell cycle and targets the expression of Olig2 in Glioma Stem like Cells

Glioma Stem-like Cells (GSCs) isolated from patient derived tumors have high metabolic activity and survive in the absence of exogenous growth factors. We recently demonstrated that acute D609 (Tricyclodecan-9-yl-xanthogenate), a PC-PLC inhibitor with anti-oxidative property, can decrease the ATP content & GADD45β protein in GSCs cultured without growth factors, but not in the presence of growth factors. In this study we examined the effect of chronic D609 treatment on GSCs cultured in complete medium containing growth factors. Our results show that chronic exposure of GSCs to D609 decreased the ATP content and reduced the expression of GADD45β protein. Furthermore, cyclin D1 content and the phosphorylation of retinoblastoma protein also diminished, resulting in the arrest of cells in G1 phase of cell cycle after D609 treatment. In addition, the expression of Olig2, a protein responsible for the progression of glioblastoma was reduced by D609. Together these results indicate that chronic D609 treatment can inhibit the growth of glioma cells by arresting cells in G1 phase of cell cycle and/or reducing Olig2 expression.

CD44 Interacts with HIF-2α to Modulate the Hypoxic Phenotype of Perinecrotic and Perivascular Glioma Cells

Hypoxia-inducible factors enhance glioma stemness, and glioma stem cells have an amplified hypoxic response despite residing within a perivascular niche. Still, little is known about differential HIF regulation in stem versus bulk glioma cells. We show that the intracellular domain of stem cell marker CD44 (CD44ICD) is released at hypoxia, binds HIF-2α (but not HIF-1α), enhances HIF target gene activation, and is required for hypoxia-induced stemness in glioma. In a glioma mouse model, CD44 was restricted to hypoxic and perivascular tumor regions, and in human glioma, a hypoxia signature correlated with CD44. The CD44ICD was sufficient to induce hypoxic signaling at perivascular oxygen tensions, and blocking CD44 cleavage decreased HIF-2α stabilization in CD44-expressing cells. Our data indicate that the stem cell marker CD44 modulates the hypoxic response of glioma cells and that the pseudo-hypoxic phenotype of stem-like glioma cells is achieved by stabilization of HIF-2α through interaction with CD44, independently of oxygen.

Metabolic regulation of glioma stem-like cells in the tumor micro-environment

Cancer metabolism has emerged as one of the most interesting old ideas being revisited from a new perspective. In the early 20th century Otto Warburg declared metabolism the prime cause in a disease of many secondary causes, and this statement seems more prescient in view of modern expositions into the true nature of tumor evolution. As the complexity of tumor heterogeneity becomes more clear from a genetic perspective, it is important to consider the inevitably heterogeneous metabolic components of the tumor and the tumor microenvironment. High grade gliomas remain one of the most difficult to treat solid tumors, due in part to the highly vascularized nature of the tumor and the maintenance of more resistant stem-like subpopulations within the tumor. Maintenance of glioma stem cells (GSCs) requires specific alterations within the cells and the greater tumor microenvironment with regards to signaling and metabolism. Specific niches within gliomas help foster the survival of stem-like sub-populations of cells with high tumorigenicity and high metabolic plasticity. Understanding these maintenance pathways and the metabolic dependencies within the niche may highlight potential avenues of addressing tumor resistance and recurrence in glioma patients.

The Impact of the Tumor Microenvironment on the Properties of Glioma Stem-Like Cells.

Glioblastoma is the most common and highly malignant primary brain tumor, and patients affected with this disease exhibit a uniformly dismal prognosis. Glioma stem-like cells (GSCs) are a subset of cells within the bulk tumor that possess self-renewal and multi-lineage differentiation properties similar to somatic stem cells. These cells also are at the apex of the cellular hierarchy and cause tumor initiation and expansion after chemo-radiation. These traits make them an attractive target for therapeutic development. Because GSCs are dependent on the brain microenvironment for their growth, and because non-tumorigenic cell types in the microenvironment can influence GSC phenotypes and treatment response, a better understanding of these cell types is needed. In this review, we provide a focused overview of the contributions from the microenvironment to GSC homing, maintenance, phenotypic plasticity, and tumor initiation. The interaction of GSCs with the vascular compartment, mesenchymal stem cells, immune system, and normal brain cell types are discussed. Studies that provide mechanistic insight into each of these GSC–microenvironment interactions are warranted in the future.

Abstract 5427: Distinctions between the metabolic changes in glioblastoma cells and glioma stem-like cells following irradiation

Abstract Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults and, even with aggressive treatment that includes surgical resection, radiation, and temozolomide administration, prognosis is poor due to tumor recurrence. There is evidence that within GBMs a small number of glioma stem- like cells (GSCs) exist, which are thought to be radiation resistant and may be capable of repopulating a tumor after treatment. Like most cancers, GBMs largely employ aerobic glycolysis to create ATP, a phenomenon known as the Warburg effect. Experiments have shown that the cellular metabolism of GSCs differs from that of differentiated cells, making them an attractive target for novel therapeutic approaches. Much work has been done to analyze the metabolic profiles of GSCs with the goal of identifying potential therapeutic targets, but little data exists linking metabolic changes to radiation resistance. The purpose of this study is to characterize the metabolic differences between glioma stem- like cells and traditional GBM tumor cells with and without radiation treatment (IR). To this end, we compared the metabolism of a human derived GSC line with two commonly used GBM cell lines before and after IR. At baseline, we find that glioma stem-like cells are more quiescent than GBM cells, which have higher levels of both glycolysis and oxidative phosphorylation. GBM lines show higher levels of both basal and maximal respiration, as well as basal glycolysis and glycolytic capacity than GSCs. They also express higher levels of energy, glycolysis, and TCA cycle metabolites than GSCs. Inversely, GSCs demonstrate metabolic signs of quiescence such as decreased NEAA synthesis. After IR, the radiation- sensitive GBM tumor cell line (U251) exhibits increases in all metabolic pathways, whereas the levels of glycolytic and oxidative metabolites in the GSCs remain unchanged. All cell lines show an increase of ATP and NAD production following IR. These findings indicate that the metabolism of GSCs undergoes different alterations than that of GBM tumor cells after IR, making them an attractive target for novel therapeutic approaches in conjunction with radiation therapy. Additionally, differences in metabolic signatures between GSC lines could be useful for non-invasive diagnostic modalities such as 13C MR spectroscopy. Citation Format: Elizabeth I. Spehalski, Cord Peters, Philip Tofilon, Kevin Camphausen. Distinctions between the metabolic changes in glioblastoma cells and glioma stem-like cells following irradiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5427. doi:10.1158/1538-7445.AM2017-5427

Abstract 5009: Self-renewal of hypoxic glioma stem-like cells is facilitated by secretion-mediated STAT3 activation

Abstract Glioblastoma, a type of high-grade glioma, is the most common and the most aggressive primary brain tumor of adults. The median survival is 12-15 months. Despite multimodality treatment, most high-grade gliomas eventually recur and are ultimately incurable. Several studies suggest that the initiation, progression, and recurrence of gliomas are driven at least partly by cancer stem-like cells. A defining characteristic of these cancer stem-like cells is their capacity to self-renew. We have identified a pathway dependent upon tumor hypoxia, a commonly found pathologic feature of GBM, that promotes self-renewal through the HIF1α-JAK1/2-STAT3 axis. Under hypoxic conditions, the expression of HIF1α is greatly increased in GBM stem-like cells leading to the activation of JAK1/2-STAT3 which mediates enhanced tumor stem-like cell self-renewal. Our data further demonstrate the importance of VEGF secretion for this pathway of hypoxia-mediated self-renewal. Using tumor stem-like cells derived from the S100β-v-erbB/p53-/- mouse model of spontaneous high-grade glioma, we have discovered that the activation of STAT3 through hypoxia-mediated VEGF secretion is required for self-renewal of glioma stem-like cells. Media conditioned by these S100β-v-erbB/p53-/- tumor sphere cells cultured under hypoxic conditions promoted self-renewal in S100β-v-erbB/p53-/- glioma stem-like cells and depletion of VEGF from this media was sufficient to inhibit STAT3 phosphorylation. Moreover, we have been able to identify two drugs: Brefeldin A and EHT-1864, that significantly inhibit VEGF secretion, decrease stem cell self-renewal, inhibit tumor growth, and increase the survival of mice allografted with S100β-v-erbB/p53-/- glioma stem-like cells. Cells treated with these drugs express molecular signatures that correlated strongly with the increased survival of GBM patients. These findings suggest a novel treatment strategy to inhibit hypoxia-mediated self-renewal of glioma stem-like cells in high-grade gliomas by targeting the secretion of extracellular, paracrine mediators of self-renewal. Citation Format: Damian A. Almiron Bonnin, Matthew C. Havrda, Huan Liu, Myung Chang Lee, Matthew H. Ung, Chao Cheng, Mark A. Israel. Self-renewal of hypoxic glioma stem-like cells is facilitated by secretion-mediated STAT3 activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5009. doi:10.1158/1538-7445.AM2017-5009

Abstract 1174: CT179 degrades the olig2 transcription factor in glioblastoma stem-like cells and prolongs survival

Abstract Olig2 is a bHLH transcription factor that has been shown to be a key driver of glioblastoma (GBM) tumorigenesis. Olig2 is expressed in human tumor-derived glioma stem-like cells. A growing body of evidence suggests that glioma stem-like cells (GSCs) are more representative of their parent tumors when cultured under defined serum-free conditions with the mitogens epidermal growth factor (EGF) and fibroblast growth factor (FGF). Furthermore, recent research has indicated that human Proneural GSCs grown in vitro in PDGF are more tumorigenic in vivo. We have designed and synthesized a potent inhibitor of Olig2 that possesses all the necessary properties for clinical development as an important new GBM therapeutic. CT179 dose dependently causes the degradation of Olig2 in multiple GBM cell lines and this correlates to G2/M arrest and increased apoptosis. CT179 also induces the loss of expression of EGFR and PDGFR in human GSCs. CT179 achieves high concentrations in the CNS with a long duration of pharmacologic action. Furthermore, CT179 significantly extends survival of mice implanted orthotopically with patient-derived human tumor GSCs. Immunohistochemistry demonstrates a marked reduction of Olig2 positive cells in the tumor bearing animals. Together, these results indicate that CT179 has significant promise as a new therapeutic for the treatment of human GBM. Citation Format: Gordon R. Alton, Graham Beaton, Susan Knowles, Gregory Stein, Santosh Kesari. CT179 degrades the olig2 transcription factor in glioblastoma stem-like cells and prolongs survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1174. doi:10.1158/1538-7445.AM2017-1174

Abstract 3348: UniD: unified and integrated diagnostic pipeline for malignant gliomas based on DNA methylation data

Abstract Prognostic and predictive molecular diagnostics for patients with gliomas typically rely on multiple assays, requiring large amounts of tissue and high cost. DNA methylation has been utilized to identify prognostic subsets and high-throughput platforms exist that are suitable for archival tissue. Therefore, we developed a unified and integrated diagnostic pipeline that can assess multiple prognostic and predictive biomarkers using only the Illumina Infinium Methylation array. This pipeline includes two parts: data processing and diagnostic biomarkers. Data processing starts from the raw data and followed with quantitative sample, probe, and batch quality control. The diagnostic biomarkers include a glioma methylation assay that predicts radiation response (GaMA); tumor classification enriched for TCGA expression subclasses; copy number alterations including phosphatase and tensin homolog (PTEN) loss, epidermal growth factor receptor (EGFR) amplification, and chromosomes 1p/19q co-deletion; CpG island methylation phenotype (G-CIMP); isocitrate dehydrogenase (IDH) mutation, and O6-methylguanine-DNA methyltransferase (MGMT) promoter hypermethylation. WHO grade II-IV gliomas were analyzed in both publically available and institutional datasets. A signature was identified to effectively distinguished radiation resistant from radiation sensitive glioma stem-like cells (GSCs). Signature has been applied to 272 TCGA GBM samples from patients who received standard radiotherapy (RT). The survival analysis showed that the subgroup with RT-sensitive and RT-resistant have significant difference in survival time (log-rank test p-value = 0.0016). Gene expression subclasses prediction biomarker was build by using the revised TCGA gene expression subclasses as gold standard. The prediction accuracy in test data set was 83.5% in the homogeneous subgroup, 71.0% in the semi-heterogeneous subgroup, and 62.1% in the heterogeneous subgroup. The prediction accuracy decreased as tumor heterogeneity increased. Certain copy number alteration events were predicted by developing specific signatures. Revised methylation signatures were developed for IDH mutation and G-CIMP status respectively, which can identified 99% of those samples. 230 GBM samples with 450k data available were tested with MGMT methylation-specific real-time PCR for MGMT methylation status. The methylation-based MGMT prediction accuracy reached about 90%. In summary, we have developed a single, FFPE-based pipeline for unified and integrated determination of multiple biomarkers of malignant glioma. Citation Format: Jie Yang, Qianghu Wang, Lihong Long, Ravesanker Ezhilarasan, Erik Sulman. UniD: unified and integrated diagnostic pipeline for malignant gliomas based on DNA methylation data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3348. doi:10.1158/1538-7445.AM2017-3348

Abstract 1125A: β-hydroxybutyrate inhibits histone deacetylase activity and radiosensitizes malignant glioma cells

Abstract Several preclinical studies support the use of the therapeutic ketogenic diet (KD) and/or caloric restriction (CR) as an adjuvant to cancer therapy. This data not only suggests that this approach alters multiple hallmarks of cancer growth, but that it may also enhance other therapeutic modalities. We have demonstrated that the unrestricted KD greatly increases survival in a mouse model of malignant glioma when administered in combination with radiation, yet the underlying mechanisms are not fully elucidated. Mounting evidence suggests that the abnormal epigenetic landscape in tumors may, among other things, impact radiosensitivity. Specifically, many cancers exhibit increased histone deacetylase (HDAC) activity which contributes to the epigenetic milieu found in tumors and alters DNA damage repair. It was recently demonstrated that the ketone body β-hydroxybutyrate (BHB) inhibits HDAC activity in normal mouse tissue. As the use of HDAC inhibitors as anti-cancer agents has generated great interest, we examined the effect of BHB on HDAC activity and radiosensitivity in the context of malignant glioma. We found that BHB radiosensitizes mouse glioma, human glioma and human glioma stem-like cells in vitro. We also demonstrate that BHB inhibits HDAC activity in a dose dependent manner and alters key components of DNA damage repair. Taken together this data opens up another avenue for understanding the mechanisms underlying the KD and its impact on radiation therapy, ultimately helping us better understand how to implement it for clinical use in oncology. Citation Format: Eric C. Woolf, Alex P. Rossi, Helena B. Silva-Nichols, Kara D. Gardner, Nelofer Syed, Adrienne C. Scheck. β-hydroxybutyrate inhibits histone deacetylase activity and radiosensitizes malignant glioma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1125A. doi:10.1158/1538-7445.AM2017-1125A

The Expression of Connexins and SOX2 Reflects the Plasticity of Glioma Stem-Like Cells

Glioblastoma (GBM) is the most malignant primary brain tumor, with an average survival rate of 15 months. GBM is highly refractory to therapy, and such unresponsiveness is due, primarily, but not exclusively, to the glioma stem-like cells (GSCs). This subpopulation express stem-like cell markers and is responsible for the heterogeneity of GBM, generating multiple differentiated cell phenotypes. However, how GBMs maintain the balance between stem and non-stem populations is still poorly understood. We investigated the GBM ability to interconvert between stem and non-stem states through the evaluation of the expression of specific stem cell markers as well as cell communication proteins. We evaluated the molecular and phenotypic characteristics of GSCs derived from differentiated GBM cell lines by comparing their stem-like cell properties and expression of connexins. We showed that non-GSCs as well as GSCs can undergo successive cycles of gain and loss of stem properties, demonstrating a bidirectional cellular plasticity model that is accompanied by changes on connexins expression. Our findings indicate that the interconversion between non-GSCs and GSCs can be modulated by extracellular factors culminating on differential expression of stem-like cell markers and cell-cell communication proteins. Ultimately, we observed that stem markers are mostly expressed on GBMs rather than on low-grade astrocytomas, suggesting that the presence of GSCs is a feature of high-grade gliomas. Together, our data demonstrate the utmost importance of the understanding of stem cell plasticity properties in a way to a step closer to new strategic approaches to potentially eliminate GSCs and, hopefully, prevent tumor recurrence.

LPS alters the immuno-phenotype of glioma and glioma stem-like cells and induces in vivo antitumor immunity via TLR4

BackgroundThis study examined the ability of lipopolysaccharide (LPS) to affect glioma and glioma stem-like cells (GSCs) in vitro and to induce antitumor immunity in vivo and the role of TLR4 in these processes.MethodsUsing RT-PCR and immunohistochemistry, we examined the expression of TLR4 in 34 glioblastoma clinical samples. Using real time-PCR, western blot and ELISA analyses, the effect of LPS stimulation on the expression of immune related molecules was evaluated in RG2 and U87 GSCs. Control or LPS-pretreated RG2 GSCs were intracranially or subcutaneously implanted into wild-type or nude Fisher 344 rats. Histopathological examinations were used to assess tumor progression and immune infiltration and Kaplan-Meier analyses to compare survival times of the animal models.ResultsTLR4 was highly expressed in glioblastoma clinical samples. In vitro LPS stimulation for 6 h significantly altered expression of immune related molecules in RG2 and U87 GSCs. However, prolonged LPS stimulation diminished this effect. Rats inoculated intracranially with LPS-pretreated RG2 GSCs survived significantly longer than rats inoculated with control RG2 GSCs. In vivo, LPS-pretreated RG2 GSCs expressed higher levels of MHC molecules, CXCL10 and TNF-α and recruited more CD8+ lymphocytes. However, intratumoral LPS treatment was not equally beneficial. Furthermore, the in vitro and in vivo effects of LPS stimulation appeared to be largely TLR4-dependent.ConclusionLPS pretreatment promotes the recognition and eradication of tumor GSCs in vivo when the immune function of the tumor-bearing host is intact. In addition, our data indicate a complex relationship between bacterial infection and glioma prognosis.

Molecular and Microenvironmental Determinants of Glioma Stem-Like Cell Survival and Invasion.

Glioblastoma multiforme (GBM) is the most frequent primary brain tumor in adults with a 5-year survival rate of 5% despite intensive research efforts. The poor prognosis is due, in part, to aggressive invasion into the surrounding brain parenchyma. Invasion is a complex process mediated by cell-intrinsic pathways, extrinsic microenvironmental cues, and biophysical cues from the peritumoral stromal matrix. Recent data have attributed GBM invasion to the glioma stem-like cell (GSC) subpopulation. GSCs are slowly dividing, highly invasive, therapy resistant, and are considered to give rise to tumor recurrence. GSCs are localized in a heterogeneous cellular niche, and cross talk between stromal cells and GSCs cultivates a fertile environment that promotes GSC invasion. Pro-migratory soluble factors from endothelial cells, astrocytes, macrophages, microglia, and non-stem-like tumor cells can stimulate peritumoral invasion of GSCs. Therefore, therapeutic efforts designed to target the invasive GSCs may enhance patient survival. In this review, we summarize the current understanding of extrinsic pathways and major stromal and immune players facilitating GSC maintenance and survival.

Correction: Dedifferentiation of Glioma Cells to Glioma Stem-like Cells By Therapeutic Stress-induced HIF Signaling in the Recurrent GBM Model.

Correction: Dedifferentiation of Glioma Cells to Glioma Stem-like Cells By Therapeutic Stress-induced HIF Signaling in the Recurrent GBM Model.

Targeting myeloid derived suppressor cells: Phase 0/1 trial of low dose capecitabine + bevacizumab in patients with recurrent glioblastoma.

e13507 Background: Glioblastoma (GBM) creates an immunosuppressive environment that allows tumor growth. Myeloid derived suppressor cells (MDSCs), a heterogeneous class of immunosuppressive cells, mediate immune suppression in GBMs. MDSCs are up-regulated in the blood of patients with GBM and multiple other tumor types. We have developed a novel strategy to target GBM immunosuppression using low dose 5-fluorouracil (5-FU) that, targets immune cells and does not depend on blood brain or blood tumor barrier penetration. Marked MDSC depletion occurs at 5-FU doses in mice equivalent to < 10% of the normal human dosing. Goal: proof of concept that MDSC suppression in feasible in GBM pts with low-dose capecitabine [cap], an oral 5-FU analogue). Methods: Eligibility: Recurrent GBM in need of surgical resection; no prior cap or bevacizumab (bev). Cohorts of 3-6 patients receive low-dose cap 150 mg/m2/d (dose level [DL] 1) for 7 days before surgery. After surgery, patients resume cap for one cycle after which bev is added. Blood MDSC, immune cell levels, and relevant secreted factors are measured at baseline; pre- and post-op; and after the addition of bev. Tumors are assayed for MDSCs and glioma stem-like cells (GSCs). Primary endpoint: MDSC and T-Regulatory cell reduction after cap. Secondary endpoints: Tumor concentrations of MDSCs, GSCs, and T-reg cells; safety; and PFS6. Results: Three of the 4 patients enrolled have data available. All patients received 5 days of pre-op cap at DL 1 with MDSC reductions of 20, 26, and 79% from baseline. The first patient reached a reduction of 93% (measured 2 days after pre-op course) whereas the other 2 experienced return to baseline. The regulatory T cells (T-reg) also fell approximately 15, 20, and 50%, respectively. CD8 concentrations appeared to rise at the time of MDSC and T-reg reduction. No patient experienced grade 3 or higher toxicity. Conclusions: Low dose capecitabine appears to reduce MDSC concentrations with minimal toxicity. During this course T-regs also fell and CD8 concentrations rose. Dose escalation continues. (NCT02669173) (Supported by Musella Foundation, Blast GBM, Sontag Foundation, Velosano, Mylan Pharmaceuticals) Clinical trial information: NCT02669173.

MDM2 Degrades Deacetylated Nucleolin Through Ubiquitination to Promote Glioma Stem-Like Cell Enrichment for Chemotherapeutic Resistance.

Glioblastoma multiforme (GBM) is the most fatal of all brain cancers, and the standard care protocol for GBM patients is surgical tumor resection followed by radiotherapy and temozolomide (TMZ)-mediated chemotherapy. However, tumor recurrence frequently occurs, and recurrent GBM exhibits more malignancy and less sensitivity in response to chemotherapy. The malignancy and drug resistance primarily reflect the small population of glioma stem-like cells (GSC). Therefore, understanding the mechanism that controls GSC enrichment is important to benefit the prognosis of GBM patients. Nucleolin (NCL), which is responsible for ribosome biogenesis and RNA maturation, is overexpressed in gliomas. However, the role of NCL in GSC development and drug resistance is still unclear. In this study, we demonstrate that NCL attenuated GSC enrichment to enhance the sensitivity of GBM cells in response to TMZ. In GSC enrichment, NCL was significantly reduced at the protein level as a result of decreased protein stability. In particular, the inhibition of HDAC activity by suberoylanilide hydroxamic acid rescued NCL acetylation accompanied by the loss of mouse double minute 2 homolog (MDM2)-mediated ubiquitination. In addition, we found that NCL ubiquitination resulted from the activation of STAT3- and JNK-mediated signaling in GSC. Moreover, NCL inhibited the formation of stem-like spheres by attenuating the expression of Sox2, Oct4, and Bmi1. Furthermore, NCL sensitized the response of GBM cells to TMZ. Based on these findings, NCL expression is a potential indicator to predict chemotherapeutic efficiency in GBM patients.

IGF2 mRNA binding protein 3 (IMP3) promotes glioma cell migration by enhancing the translation of RELA/p65

The diffusely infiltrative nature of glioblastoma (GBM) makes them highly recurrent. IGF2 mRNA-binding protein 3 (IMP3), a GBM upregulated RNA binding protein, promotes glioma cell migration. An integrative bioinformatics analysis identified p65 (RELA), a subunit of NF-κB heterodimer as a target and an important mediator of IMP3 promoted glioma cell migration. IMP3 increased p65 protein levels without any change in p65 transcript levels, but promoted its polysome association. RIP-PCR demonstrated the binding of IMP3 to p65 transcript. UV crosslinking experiments with in vitro transcribed RNA confirmed the specific and direct binding of IMP3 to sites on p65 3′UTR. Further, IMP3 induced luciferase activity from p65 3′UTR reporter carrying wild type sites but not mutated sites. Exogenous overexpression of p65 from a 3′UTR-less construct rescued the reduced migration of glioma cells in IMP3 silenced condition. In addition, IMP3 silencing inhibited glioma stem-like cell maintenance and migration. The exogenous overexpression of 3′UTR-less p65 significantly alleviated the inhibition of neurosphere formation observed in IMP3 silenced glioma stem-like cells. Further, we show that IMP3 is transcriptionally activated by NF-κB pathway indicating the presence of a positive feedback loop between IMP3 and p65. This study establishes p65 as a novel target of IMP3 in increasing glioma cell migration and underscores the significance of IMP3-p65 feedback loop for therapeutic targeting in GBM.

NSPc1 promotes cancer stem cell self-renewal by repressing the synthesis of all-trans retinoic acid via targeting RDH16 in malignant glioma.

Polycomb group (PcG) proteins play an important role in development and stem cell maintenance, and their dysregulation have been closely linked to oncogenesis and cancer stem cell phenotypes. Here, we found that nervous system polycomb 1 (NSPc1) was highly expressed in stem cell-like glioma cells (SLCs). Knockdown of NSPc1 in SLCs resulted in impaired neurosphere formation and self-renewal abilities, down-regulated expression of stemness markers such as NESTIN, CD133 and SOX2, and decreased capacity to propagate subcutaneous xenografts. In contrast, glioma cells overexpressing NSPc1 exhibited a stem cell-like phenotype, up-regulated expression of stemness markers NESTIN, CD133 and SOX2, and an enhanced capacity to propagate subcutaneous xenografts. Furthermore, we identified that NSPc1 epigenetically repressed the expression of retinol dehydrogenase 16 (RDH16) by directly binding to a region upstream (-1073 to -823) of the RDH16 promoter. Next, we confirmed that RDH16 is a stemness suppressor that partially rescues SLCs from the NSPc1-induced increase in neurosphere formation. Finally, we showed that ATRA partly reversed the NSPc1-induced stemness enhancement in SLCs, through mechanisms correlated with an ATRA-dependent decrease in the expression of NSPc1. Thus, our results demonstrate that NSPc1 promotes cancer stem cell self-renewal by repressing the synthesis of ATRA via targeting RDH16 and may provide novel targets for glioma treatment in the future.

Ionizing radiation augments glioma tropism of mesenchymal stem cells.

OBJECTIVE Mesenchymal stem cells (MSCs) have been shown to localize to gliomas after intravascular delivery. Because these cells home to areas of tissue injury, the authors hypothesized that the administration of ionizing radiation (IR) to tumor would enhance the tropism of MSCs to gliomas. Additionally, they sought to identify which radiation-induced factors might attract MSCs. METHODS To assess the effect of IR on MSC migration in vitro, transwell assays using conditioned medium (CM) from an irradiated commercially available glioma cell line (U87) and from irradiated patient-derived glioma stem-like cells (GSCs; GSC7-2 and GSC11) were employed. For in vivo testing, green fluorescent protein (GFP)-labeled MSCs were injected into the carotid artery of nude mice harboring orthotopic U87, GSC7-2, or GSC17 xenografts that were treated with either 0 or 10 Gy of IR, and brain sections were quantitatively analyzed by immunofluorescence for GFP-positive cells. These GSCs were used because GSC7-2 is a weak attractor of MSCs at baseline, whereas GSC17 is a strong attractor. To determine the factors implicated in IR-induced tropism, CM from irradiated GSC7-2 and from GSC11 was assayed with a cytokine array and quantitative ELISA. RESULTS Transwell migration assays revealed statistically significant enhanced MSC migration to CM from irradiated U87, GSC7-2, and GSC11 compared with nonirradiated controls and in a dose-dependent manner. After their intravascular delivery into nude mice harboring orthotopic gliomas, MSCs engrafted more successfully in irradiated U87 (p = 0.036), compared with nonirradiated controls. IR also significantly increased the tropism of MSCs to GSC7-2 xenografts (p = 0.043), which are known to attract MSCs only poorly at baseline (weak-attractor GSCs). Ionizing radiation also increased the engraftment of MSCs in strong-attractor GSC17 xenografts, but these increases did not reach statistical significance. The chemokine CCL2 was released by GSC7-2 and GSC11 after irradiation in a dose-dependent manner and mediated in vitro transwell migration of MSCs. Immunohistochemistry revealed increased CCL2 in irradiated GSC7-2 gliomas near the site of MSC engraftment. CONCLUSIONS Administering IR to gliomas enhances MSC localization, particularly in GSCs that attract MSCs poorly at baseline. The chemokine CCL2 appears to play a crucial role in the IR-induced tropism of MSCs to gliomas.