Glioma Maintenance Research Articles

Inhibitors of CK2 Kinase for the Treatment of Gliomas and Other Brain Tumors

Protein kinase CK2 has become a target of experimental antiglioma therapies as laboratory data are almost uniformly favorable and the number of synthetized CK2 inhibitors is rapidly growing. The evidence of their use for other brain tumors is on the increase as well. Great expectations are entrusted in naturally occurring compounds capable of inhibiting CK2 kinase. These compounds are extracted and purified by means of biochemistry methods and are amenable for innovative drug delivery systems as well. CK2 kinase inhibitors have been proven suitable for combined therapies with other investigational antiglioma agents and treatment modalities. However a greater share of efforts should be undertaken towards inhibiting functions relatively specific for glial tumors including infiltrative growth and invasiveness or maintenance of glioma initiating cells. Many of these function appear to converge on mTOR and JAK/STAT pathways which are being meticulously studied in this respect. Protein kinase CK2 holds therapeutic promise especially when combined with other agents aimed at molecular signatures of gliomas.

Abstract 5287: Ex vivo pharmacologic inhibition of STAT3 effectively targets tumor and tumor associated suppressor cells in high-grade glioma 3D spheroids

Abstract Gliomas, a type of brain tumor originating from glial cells, comprise a heterogenous group of neoplasms. High-grade gliomas, in particular, present significant therapeutic challenges in the clinic. Unlike other solid tumors, the heterogenous glioma microenvironment is heavily enriched with tumor associated suppressor cells including myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), which can account for 30-40% of the cellular composition1. These pro-tumorigenic myeloid cells are thought to contribute to the resistance of glioma to immunotherapy. Efforts to target the suppressive nature of these dominating cell types are in development to yield more therapeutic options for patients with these devastating diseases. Constitutive activation of STAT3 in gliomas has been associated with increased tumor progression and reduced immune response. We have observed in other cancer models that a phenotypic switch occurs from STAT3 pathway activation in 3D spheroid culture to STAT3 inactivation when cultured in 2D. Therefore, we sought to determine the role of STAT3 activation and its role in the interplay between glioma and their immune suppressive environment using a 3D spheroid model. We modified our commercially available test platform, 3D-Predict™ Glioma, such that it is conducive to monitoring immune cell function. Newly diagnosed treatment naive primary samples from HGG patients were dissociated and cryogenically preserved and banked. Samples were profiled for the abundance of tumor associated suppressive myeloid cells using flow cytometry. We determined that relative proportions of tumor suppressor cells were detectable and varied across HGG primary patient samples with TAMs being the predominate immune cell type identified. Pro-tumorigenic TAM markers including CD163 and CD206 were correlated with temozolomide response and were associated with poor patient prognosis. When we evaluated the role of p-STAT in HGG primary samples, p-STAT was detected at baseline and abundance was related back to the presence of tumor associated immune suppressor cells. The impact of STAT3 signaling blockade by the inhibitor WP1066 was determined ex vivo, and efficacy was related back to MGMT promotor methylation status and patient clinical outcome. We detected shifts in cytokine signatures following treatment of HGG 3D spheroid models with WP1066 which are known to target tumor associated suppressor cell functions. This work demonstrates the critical role suppressive immune cells play in HGG clinical response. Given p-STAT can be altered in 2D culture compared to 3D culture, ex vivo therapeutic response profiling of HGG samples is necessary using the more physiologically relevant spheroid culture model. 1.Hambardzumyan et al. The role of microglia and macrophages in glioma maintenance and progression. Nat. Neurosci. 19, 20-27 (2016). Citation Format: Katy A. Lassahn, Ashley K. Elrod, Teresa M. DesRochers, Kathryn M. Appleton. Ex vivo pharmacologic inhibition of STAT3 effectively targets tumor and tumor associated suppressor cells in high-grade glioma 3D spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5287.

Biomimetic neutrophil and macrophage dual membrane-coated nanoplatform with orchestrated tumor-microenvironment responsive capability promotes therapeutic efficacy against glioma

Malignant glioma is a significant problem in public health worldwide, due to high infiltrative glioma growth, chemotherapeutic tumor resistance, and formidable obstacle of blood–brain barrier (BBB). Therapeutic drug delivery platforms that can overcome the BBB hurdle and target glioma are urgently needed. Encouraged by the involvement of immune cells in inflammatory response, and their important role in regulating glioma maintenance and progression, we introduce an effective approach based on neutrophil-macrophage hybrid membrane coating nanotechnology for glioma treatment. By covering a rapamycin (RAPA)-loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticle with neutrophil and macrophage membranes (NMm), this biomimetic nanoparticle (NMm-PLGA/RAPA) possesses spontaneous BBB penetrating, and inherently combines the stimuli-homing responsiveness of macrophage with the inflammatory chemotaxis of neutrophils. An in vivo brain inflammatory study confirms that the NMm-PLGA/RAPA can efficiently transmigrate across BBB, respond to inflammatory stimuli, and specifically accumulate within the inflammatory site. On injection of NMm-PLGA/RAPA into a mouse glioma model, the NMm-PLGA/RAPA is found to target the brain tumor, and is capable of eliminating glioma cells and inducing durable tumor regression. Our neutrophil and macrophage membrane-coated nanoplatform presents a viable and efficient drug delivery system able to increase therapeutic efficacy for glioma and possibly other malignant tumors.

Role of Inflammatory Mediators, Macrophages, and Neutrophils in Glioma Maintenance and Progression: Mechanistic Understanding and Potential Therapeutic Applications.

Simple SummaryThe tumor microenvironment is a complex network comprised of neoplastic and a variety of immune cells, proteins, and inflammatory mediators. Previous studies have shown that during cancer progression, diverse inflammatory molecules, either directly or indirectly via recruiting immune cells, support the process of carcinogenesis. The present review focuses on the mechanistic understanding of the oncogenic role of these inflammatory mediators and immune cells, particularly tumor-associated macrophages (TAMs) and tumor-associated neutrophils (TANs) in glioma maintenance and progression. Moreover, the potential therapeutic benefits of targeting inflammatory mediators, immune cells, and associated signaling pathways in glioma genesis have also been discussed.Gliomas are the most common, highly malignant, and deadliest forms of brain tumors. These intra-cranial solid tumors are comprised of both cancerous and non-cancerous cells, which contribute to tumor development, progression, and resistance to the therapeutic regimen. A variety of soluble inflammatory mediators (e.g., cytokines, chemokines, and chemotactic factors) are secreted by these cells, which help in creating an inflammatory microenvironment and contribute to the various stages of cancer development, maintenance, and progression. The major tumor infiltrating immune cells of the tumor microenvironment include TAMs and TANs, which are either recruited peripherally or present as brain-resident macrophages (microglia) and support stroma for cancer cell expansion and invasion. These cells are highly plastic in nature and can be polarized into different phenotypes depending upon different types of stimuli. During neuroinflammation, glioma cells interact with TAMs and TANs, facilitating tumor cell proliferation, survival, and migration. Targeting inflammatory mediators along with the reprogramming of TAMs and TANs could be of great importance in glioma treatment and may delay disease progression. In addition, an inhibition of the key signaling pathways such as NF-κB, JAK/STAT, MAPK, PI3K/Akt/mTOR, and TLRs, which are activated during neuroinflammation and have an oncogenic role in glioblastoma (GBM), can exert more pronounced anti-glioma effects.

ADO/hypotaurine: a novel metabolic pathway contributing to glioblastoma development

Significant advance has been made towards understanding glioblastoma metabolism through global metabolomic profiling. However, hitherto little is known about the role by which altered metabolism plays in driving the aggressive glioma phenotype. We have previously identified hypotaurine as one of the top-ranked metabolites for differentiating low- and high-grade tumors, and that there is also a strong association between the levels of intratumoral hypotaurine and expression of its biosynthetic enzyme, cysteamine (2-aminoethanethiol) dioxygenase (ADO). Using transcription profiling, we further uncovered that the ADO/hypotaurine axis targets CCL20 secretion through activating the NF-κB pathway to drive the self-renewal and maintenance of glioma ‘cancer stem cells’ or glioma cancer stem-like cells. Conversely, abrogating the ADO/hypotaurine axis using CRISPR/Cas9-mediated gene editing limited glioblastoma cell proliferation and self-renewal in vitro and tumor growth in vivo in an orthotopical mouse model, indicating that this metabolic pathway is a potential key therapeutic target. Collectively, our results unveil a targetable metabolic pathway, which contributes to the growth and progression of aggressive high-grade gliomas, as well as a novel predictive marker for glioblastoma diagnosis and therapy.

PDIA4 Correlates with Poor Prognosis and is a Potential Biomarker in Glioma.

PurposeGliomas, characterized by aggressiveness and invasiveness, remain incurable after conventional therapies. The molecular mechanisms driving the progression and maintenance of glioma are still poorly understood.MethodsThe TCGA and CGGA databases were chosen for bioinformatics analysis. Gene expression profiling interactive analysis (GEPIA) was performed for differential analysis. The Kaplan–Meier method was chosen for survival analysis. Analysis of stromal and immune infiltration was performed using the ESTIMATE algorithm and xCell package. qPCR and Western blotting were performed to measure the expression of PDIA4 at the mRNA and protein levels. IHC was performed to detect the expression of PDIA4 in glioma tissues. The viability of glioma cells was evaluated by the CCK8 assay.ResultsIn this study, we identified high PDIA4 expression in gliomas that correlated with poor prognosis. The association between IDH1 and different glioma patterns also indicated the potential biological role of PDIA4 in tumor development. Mechanistically, PDIA4 interacted with multiple immunological components to promote an immunosuppressive tumor microenvironment (TME). Knockdown of PDIA4 significantly impaired the proliferation of GBM cells.ConclusionOur results confirm that PDIA4 is an efficient biomarker of gliomas, with clinical implications for prognosis and therapeutic strategies.

TMOD-29. A MOUSE MODEL OF FGFR3-TACC3 UNCOVERS MECHANISMS OF GLIOMAGENESIS AND MAINTENANCE

Abstract Glioblastoma harboring Fibroblast Growth Factor Receptor 3 (FGFR3)-Transforming Acidic Coiled-Coil containing 3 (TACC-3) (FGFR3-TACC3) fusion is a distinct molecular subclass of tumors that can be targeted by FGFR kinase inhibitors. We generated a knock-in mouse in which the expression of FGFR3-TACC3 is doxycycline inducible and reversible. The tetO-FGFR3-TACC3-IRES-GFP cassette was placed into the Rosa26 locus and the FGFR3-TACC3 knock-in strain was intercrossed with GFAP-rtTA*M2 mice to trigger expression of the tetracycline transactivator (and the fusion gene) from the GFAP promoter. To recapitulate the genetic lesions that are more frequently associated with FGFR3-TACC3 in human glioblastoma, we also intercrossed the tetO-FGFR3-TACC3-IRES-GFP;GFAP-rtTA*M2 mice with TP53+/-or Ink4A+/-mouse strains. Both strains developed aggressive and infiltrating gliomas within 90 days after induction of FGFR3-TACC3 by doxycycline containing diet. Tumors occurred with a prevalence of ~50% and were found almost exclusively at two brain locations: the lateral septum and the neuro-epithelium adjacent to the fimbria of the hippocampus, thus recapitulating the restricted expression of endogenous FGFR3 in the ventral telencephalon and ganglionic eminence during development. The FGFR3-TACC3-positive glioma resembled human glioblastoma as they exhibited marked invasiveness and vascularization, elevated proliferative index and positivity for Nestin and GFAP. Rapid tumor progression caused appearance of neurological symptoms and death within few weeks. Doxycycline withdrawal from the diet of mice diagnosed with brain tumors by MRI resulted in rapid loss of FGFR3-TACC3 from glioma cells, marked inhibition of tumor growth with essentially complete glioma regression in some cases and recovery of general and neurological clinical symptoms. Our findings validate FGFR3-TACC3 as powerful oncogene that drives development of high-grade glioma from distinct areas of the brain. They also provide an accurate pre-clinical model in which glioma maintenance requires continuous expression of the fusion gene, thus supporting therapeutic targeting of the fusion protein in patients with FGFR3-TACC3-positive glioblastoma.

Recent Advances in Targeted Therapy for Glioma.

Gliomas are the most common primary malignant brain tumors, which have a universally fatal outcome. Current standard treatment for glioma patients is surgical removal followed by radiotherapy and adjuvant chemotherapy. Due to therapeutic resistance and tumor recurrence, efforts are ongoing to identify the molecules that are fundamental to regulate the tumor progression and provide additional methods for individual treatment of glioma patients. By studying the initiation and maintenance of glioma, studies focused on the targets of tyrosine kinase receptors including EGFR, PDGFR and other crucial signal pathways such as PI3K/AKT and RAS/RAF/MAPK pathway. Furthermore, recent advances in targeting immunotherapy and stem cell therapy also brought numerous strategies to glioma treatment. This article reviewed the researches focused on the advanced strategies of various target therapies for improving the glioma treatment efficacy, and discussed the challenges and future directions for glioma therapy.

The role of microglia and macrophages in glioma maintenance and progression.

There is a growing recognition that gliomas are complex tumors composed of neoplastic and non-neoplastic cells, which each individually contribute to cancer formation, progression and response to treatment. The majority of the non-neoplastic cells are tumor-associated macrophages (TAMs), either of peripheral origin or representing brain-intrinsic microglia, that create a supportive stroma for neoplastic cell expansion and invasion. TAMs are recruited to the glioma environment, have immune functions, and can release a wide array of growth factors and cytokines in response to those factors produced by cancer cells. In this manner, TAMs facilitate tumor proliferation, survival and migration. Through such iterative interactions, a unique tumor ecosystem is established, which offers new opportunities for therapeutic targeting.

RNA Sequencing of Tumor-Associated Microglia Reveals Ccl5 as a Stromal Chemokine Critical for Neurofibromatosis-1 Glioma Growth

Solid cancers develop within a supportive microenvironment that promotes tumor formation and growth through the elaboration of mitogens and chemokines. Within these tumors, monocytes (macrophages and microglia) represent rich sources of these stromal factors. Leveraging a genetically engineered mouse model of neurofibromatosis type 1 (NF1) low-grade brain tumor (optic glioma), we have previously demonstrated that microglia are essential for glioma formation and maintenance. To identify potential tumor-associated microglial factors that support glioma growth (gliomagens), we initiated a comprehensive large-scale discovery effort using optimized RNA-sequencing methods focused specifically on glioma-associated microglia. Candidate microglial gliomagens were prioritized to identify potential secreted or membrane-bound proteins, which were next validated by quantitative real-time polymerase chain reaction as well as by RNA fluorescence in situ hybridization following minocycline-mediated microglial inactivation in vivo. Using these selection criteria, chemokine (C-C motif) ligand 5 (Ccl5) was identified as a chemokine highly expressed in genetically engineered Nf1 mouse optic gliomas relative to nonneoplastic optic nerves. As a candidate gliomagen, recombinant Ccl5 increased Nf1-deficient optic nerve astrocyte growth in vitro. Importantly, consistent with its critical role in maintaining tumor growth, treatment with Ccl5 neutralizing antibodies reduced Nf1 mouse optic glioma growth and improved retinal dysfunction in vivo. Collectively, these findings establish Ccl5 as an important microglial growth factor for low-grade glioma maintenance relevant to the development of future stroma-targeted brain tumor therapies.

Akt- or MEK-mediated mTOR inhibition suppresses Nf1 optic glioma growth.

Children with neurofibromatosis type 1 (NF1) develop optic pathway gliomas, which result from impaired NF1 protein regulation of Ras activity. One obstacle to the implementation of biologically targeted therapies is an incomplete understanding of the individual contributions of the downstream Ras effectors (mitogen-activated protein kinase kinase [MEK], Akt) to optic glioma maintenance. This study was designed to address the importance of MEK and Akt signaling to Nf1 optic glioma growth. Primary neonatal mouse astrocyte cultures were employed to determine the consequence of phosphatidylinositol-3 kinase (PI3K)/Akt and MEK inhibition on Nf1-deficient astrocyte growth. Nf1 optic glioma-bearing mice were used to assess the effect of Akt and MEK inhibition on tumor volume, proliferation, and retinal ganglion cell dysfunction. Both MEK and Akt were hyperactivated in Nf1-deficient astrocytes in vitro and in Nf1 murine optic gliomas in vivo. Pharmacologic PI3K or Akt inhibition reduced Nf1-deficient astrocyte proliferation to wild-type levels, while PI3K inhibition decreased Nf1 optic glioma volume and proliferation. Akt inhibition of Nf1-deficient astrocyte and optic glioma growth reflected Akt-dependent activation of mammalian target of rapamycin (mTOR). Sustained MEK pharmacologic blockade also attenuated Nf1-deficient astrocytes as well as Nf1 optic glioma volume and proliferation. Importantly, these MEK inhibitory effects resulted from p90RSK-mediated, Akt-independent mTOR activation. Finally, both PI3K and MEK inhibition reduced optic glioma-associated retinal ganglion cell loss and nerve fiber layer thinning. These findings establish that the convergence of 2 distinct Ras effector pathways on mTOR signaling maintains Nf1 mouse optic glioma growth, supporting the evaluation of pharmacologic inhibitors that target mTOR function in future human NF1-optic pathway glioma clinical trials.

The expression of SALL4 in patients with gliomas: high level of SALL4 expression is correlated with poor outcome.

The spalt-like transcription factor 4 (SALL4) gene has been demonstrated to be overexpressed in many malignancies, but little is known about its expression in gliomas. To explore the expression of SALL4 in patients with gliomas and the relationship between SALL4 expression and clinicopathologic characteristics, qPCR and immunohistochemical staining were used to investigate the SALL4 expression level in 54 glioma specimens and seven normal brain tissues. In vitro, siRNAs against SALL4 in U251 cell line were constructed and cell proliferation was evaluated by CCK8 assay. The SALL4 expression level in glioma was significantly higher than that in normal brain tissues (P < 0.05). Both qPCR and immunohistochemical analysis found that the expression of SALL4 was tightly correlated with glioma pathology grade (P < 0.05). Analysis using glioma and normal brain tissues revealed that SALL4 was positively proportionated to glioma cell differentiation with high sensitivity (92.59 %) and specificity (85.71 %). Survival analysis indicated the SALL4 expression was an independent prognostic factor. High level of SALL4 expression was correlated with poor outcome in patients with gliomas. This result agreed with the negative correlation between SALL4 expression and overall survival period obtaining in GBM patients from the cancer genome atlas database. The CCK8 experiments demonstrated SALL4 could significantly inhibit cell proliferation in U251 cell line (P < 0.05). The findings of the current study indicated that the SALL4 may play an important role in progression, development and maintenance of glioma.

Abstract 4321: A critical role for IGFBP2 in the progression and maintenance of glioma

Abstract Despite recent advances in detection methods and treatments for other tumor types, the standard of care for glioma patients remains relatively unchanged and survival rates remain dismal. Identification of key signaling nodes that function throughout glioma development will likely provide new targets for intervention. To elucidate the role of the critical oncogene, IGFBP2, in the progression and maintenance of glioma, we generated an inducible mouse model. This model utilizes the RCAS/Tv-a system, which places the Tv-a receptor for the avian RCAS virus under the control of the Nestin promoter to drive Tv-a expression in glial progenitors. The RCAS virus, expressing the desired oncogenes, infects Tv-a expressing cells and delivers the oncogene. By using RCAS to express a tetracycline-responsive IGFBP2 and a tetracycline transactivator, we can modify this system to generate an inducible IGFBP2 model. Using this model, we have tested the hypothesis that IGFBP2 is a key target for glioma therapy due to its important role in glioma maintenance. The studies described here aim to: (1) determine the effects of delayed IGFBP2 expression on the progression of PDGFB-induced, low-grade gliomas; and (2) determine whether continued expression of IGFBP2 is required to maintain the high-grade nature of gliomas induced by the co-expression of PDGFB and IGFBP2. Previously, our lab and others determined that over-expression of PDGFB in glial progenitors led to the development of low-grade glioma. Our lab also determined that IGFBP2 cooperates with PDGFB to promote progression to high-grade glioma. In the inducible model system, tetracycline-mediated IGFBP2 induction in vivo was confirmed by both quantitative RT-PCR of mouse brain tissue and serum ELISA, indicating that RCAS-delivered IGFBP2 is both expressed and secreted upon induction. IGFBP2 induction from birth increased the proportion of tumors that progressed to high-grade glioma, whereas delayed IGFBP2 induction led to later onset of high-grade tumor development. Significantly, there was a decrease in tumor-free survival upon IGFBP2 induction. This is consistent with analysis of transcriptome data from the Rembrandt and TCGA databases. These studies validate an important role for IGFBP2 in the progression of glioma. Ongoing studies using the inducible system are exploring the response of high-grade gliomas to sustained and brief inactivation of IGFBP2. Elucidating the roles of IGFPB2 in glioma progression and maintenance will provide critical information pertinent to the design and implementation of future therapies to target this molecule or associated signaling pathways, as well as how IGFBP2 might be useful as a marker for disease state. Further, these studies may have a more global impact on cancer research as IGFBP2 over-expression has also been associated with other high-grade tumors such as prostate and breast cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4321. doi:10.1158/1538-7445.AM2011-4321

Abstract 3311: IL6-Stat3 signaling promotes glioma stem cell (GSC) survival under hypoxia environment through inhibition of cell-death inducing gene Bnip3

Abstract Glioblastomas (GBMs) are the most common and lethal brain tumor in adults. In rapid-growing tumors like malignant glioma, hypoxia is an unavoidable condition even with aggressive angiogenesis. Recent studies implicate an important role for a restricted population of neoplastic cells (glioma stem cells, GSCs) in glioma maintenance and recurrence. However, how these glioma stem cells survive and escape from harsh conditions like hypoxia is barely known. Our previous study and others’ have shown that GSCs preferentially express the interleukin 6 (IL6) receptors and Stat3 signaling is preferentially activated in GSCs. We further found that perturbation of IL6 signaling in GSCs reduced Stat3 activation, and small molecule inhibitors of Stat3 potently induced GSC cell death partially through induction of a BCL2 family member Bnip3. Despite its controversial role in cell survival, massive expression of Bnip3 is a well-studied mediator of cell death in hypoxic tumor cells. Furthermore, we found that enhanced IL6-Stat3 signaling in GSCs could counter the induction of Bnip3 under hypoxia, which provided an extra survival advantage for these glioma stem cells under severe hypoxic condition. These data indicated that Stat3 is a downstream mediator of pro-survival IL6 signals in GSCs partially through suppressing Bnip3 expression, especially under hypoxia condition. Together, our data indicate that IL6-Stat3 signaling contributes to glioma malignancy through the promotion of GSC survival under harsh conditions like hypoxia, and that targeting IL6-Stat3 signaling pathway may offer benefit for glioma patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3311. doi:10.1158/1538-7445.AM2011-3311

Abstract 3336: Upregulated inflammatory cytokines in glioma stem cells: Targets for glioma therapies

Abstract Gliomas are the most common primary brain tumors in adults. With the highly invasive growth pattern, gliomas often escape current therapeutic modalities including combinations of surgery, radiotherapy and chemotherapy. Recent studies implicate that a minority population of glioma stem cells (GSCs) are responsible for glioma maintenance and recurrence. Interleukin (IL)-1 beta, IL-6 and IL-8 are pleiotropic proinflammatory cytokines and their excessive production in the tumor microenvironment is associated with poor disease outcome in many cancer types, including glioma. These cytokines promote tumor growth via induction of proteins associated with tumor invasion and angiogenesis and anti-apoptosis. We hypothesize that GSCs acquire their chemo-resistance and invasion by excessive cytokine production. To test the hypothesis, we compared the cytokine gene expression of a human GSC-enriched population in serum-free medium (SFM) in the presence of epidermal growth factor and fibroblast growth factor, to that of glioma cells in serum-containing medium (SCM). SFM is the neural stem/progenitor cell (NPC) medium in which NPCs proliferate in neurospheres and this medium also has been used to enrich GSCs. We found that IL-1 beta and IL-6 were upregulated in GSC-enriched population along with increased expression of Nestin, a marker of neural stem cells. More interestingly, Smad interacting protein 1 (SIP1), which is thought to be involved in glioma cell migration and invasion, was also increased in the GSC-enriched population. The paralleled upregulation of IL-1 beta and IL-6 with Nestin and SIP1 in GSC-enriched population suggest that there is a possible correlation between them. The correlation is now under investigation. Since IL-1 beta is elevated in GSC enriched population, we further determined whether IL-1 beta plays a critical role in glioma growth. Glioma cells were co-cultured with mouse NPCs expressing IL-1 receptor antagonist (IL-1ra), which can bind to IL-1 receptors without transmitting activation signals and represents a competitive inhibitor of IL-1. Proliferation of glioma cells, which were modified to express Renilla luciferase, was measured using bioluminescence imaging. Our results showed that proliferation of glioma cells was inhibited in the co-culture with NPCs expressing IL-1ra, but not in the co-culture with NPCs without expression of IL-1ra, compared to glioma cells cultured alone. The results suggest that IL-1 has an important role in glioma cell growth and NPCs engineered to express IL-1ra have therapeutic potential for gliomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3336.

Abstract C13: Targeting interleukin 6 signaling suppresses glioma stem cell survival and tumor growth

Abstract Glioblastomas are the most common and most lethal primary brain tumor. Recent studies implicate an important role for a restricted population of neoplastic cells (glioma stem cells (GSCs)) in glioma maintenance and recurrence. We now demonstrate that GSCs preferentially express two interleukin 6 (IL6) receptors: IL6 receptor alpha (IL6Ra) and glycoprotein 130 (gp130). Targeting IL6Ra or IL6 ligand expression in GSCs with the use of short hairpin RNAs (shRNAs) significantly reduces growth and neuro-sphere formation capacity while increasing apoptosis. Per-turbation of IL6 signaling in GSCs attenuates signal transducers and activators of transcription three (STAT3) activation, and small molecule inhibitors of STAT3 potently induce GSC apoptosis. These data indicate that STAT3 is a downstream mediator of prosurvival IL6 signals in GSCs. Targeting of IL6Ra or IL6 expression in GSCs increases the survival of mice bearing intracranial human glioma xenografts. IL6 is clinically significant because elevated IL6 ligand and receptor expression are associated with poor glioma patient survival. The potential utility of anti-IL6 therapies is demonstrated by decreased growth of subcutaneous human GSC-derived xenografts treated with IL6 antibody. Together, our data indicate that IL6 signaling contributes to glioma malignancy through the promotion of GSC growth and survival, and that targeting IL6 may offer benefit for glioma patients. Citation Information: Cancer Res 2009;69(23 Suppl):C13.

Activated BRAF induces gliomas in mice when combined with Ink4a/Arf loss or Akt activation

Mutations in receptor tyrosine kinase (RTK) growth factor receptors (epidermal growth factor receptor, platelet-derived growth factor receptor, MET and ERBB2), which result in downstream activation of the RAS/RAF/MEK/ERK mitogen-activated protein kinase (MAPK) pathway and PI(3)K/Akt pathway, are found in almost all high-grade gliomas and MAPK signaling is necessary for continued glioma maintenance. In addition, BRAF is mutated in the majority of low-grade gliomas and its expression and activity is significantly increased in the majority of high-grade gliomas. Although the importance of RTKs and RAS signaling in glioma development has been shown, the role of BRAF has yet to be characterized. We evaluated the effect of activated BRAF in glioma formation using the retroviral replication-competent avian leukosis virus long terminal repeat, splice acceptor (RCAS)/TVA system to transfer genes encoding activated forms of BRAF, KRas, Akt and Cre to nestin-expressing neural progenitor cells in Ink4a/Arf(lox/lox) mice in vivo. Although expression of activated BRAF alone is not sufficient for tumorigenesis, the combination of activated BRAF and Akt or BRAF with Ink4a/Arf loss is transforming. Interestingly, activated BRAF generates gliomas with characteristics similar to activated KRas in the context of Akt but not Ink4a/Arf loss. Our studies show a role for BRAF activation and signaling in glioma development and as potential target for glioma therapy.

Targeting Interleukin 6 Signaling Suppresses Glioma Stem Cell Survival and Tumor Growth

Glioblastomas are the most common and most lethal primary brain tumor. Recent studies implicate an important role for a restricted population of neoplastic cells (glioma stem cells (GSCs)) in glioma maintenance and recurrence. We now demonstrate that GSCs preferentially express two interleukin 6 (IL6) receptors: IL6 receptor alpha (IL6R alpha) and glycoprotein 130 (gp130). Targeting IL6R alpha or IL6 ligand expression in GSCs with the use of short hairpin RNAs (shRNAs) significantly reduces growth and neurosphere formation capacity while increasing apoptosis. Perturbation of IL6 signaling in GSCs attenuates signal transducers and activators of transcription three (STAT3) activation, and small molecule inhibitors of STAT3 potently induce GSC apoptosis. These data indicate that STAT3 is a downstream mediator of prosurvival IL6 signals in GSCs. Targeting of IL6R alpha or IL6 expression in GSCs increases the survival of mice bearing intracranial human glioma xenografts. IL6 is clinically significant because elevated IL6 ligand and receptor expression are associated with poor glioma patient survival. The potential utility of anti-IL6 therapies is demonstrated by decreased growth of subcutaneous human GSC-derived xenografts treated with IL6 antibody. Together, our data indicate that IL6 signaling contributes to glioma malignancy through the promotion of GSC growth and survival, and that targeting IL6 may offer benefit for glioma patients.