Migration Of Glioma Stem Cells Research Articles

SYST-24 PROPOFOL EXERTS ANTI-TUMOR EFFECTS IN GLIOMA AND THE TUMOR MICROENVIRONMENT VIA NON-CODING RNAS AND SECRETED EXOSOMES

Abstract BACKGROUND Glioblastoma (GBM), is the most common primary brain tumor. GBM contains a small subpopulation of glioma stem cells (GSCs) that are implicated in tumor recurrence and treatment resistance and therefore represent important therapeutic targets. Recent clinical studies suggest propofol impacts subsequent tumor response to treatments and patient prognosis. The effects of propofol on patient derived GSCs alone and in combination with radiation and temozolomide, (TMZ) have not been reported. Objectives: The molecular mechanisms underlying propofol’s anti-tumor effects on GSCs and its effect on cellular communication with microglia was studied. Using GSC spheroids, differentiated glioma and tumor cells on a microfluid chip, effects of propofol alone and together with radiation and TMZ on the self-renewal and stemness of GSCs, their mesenchymal transit and the proliferation and apoptosis of differentiated glioma cells was analyzed. Using transwell plates, the effects of propofol on the cross-talk of GSCs with human microglia cells was examined. RESULTS Propofol exerted a dose-dependent inhibitory effect on the self-renewal, expression of mesenchymal markers and migration of GSCs and sensitized them to both temozolomide (TMZ) and radiation. At higher concentrations propofol induced a large degree of cell death as demonstrated using microfluid chip. Propofol increased the expression of the lncRNA BDNF-AS, which acts as a tumor suppressor in GBM and silencing of this lncRNA partially abrogated propofol’s anti-tumor effects. Propofol also inhibited the pro-tumorigenic GSC-microglia cross talk via extracellular vesicles (EVs) and delivery of BDNF-AS. CONCLUSIONS Propofol exerted anti-tumor effects on GSCs and differentiated glioma cells by inhibiting cell renewal, proliferation, and mesenchymal transition and by inducing cell death at higher concentration. Propofol also sensitized GSCs to radiation and TMZ. Propofol, which is widely used in GBM surgeries, should be further explored as a potential repurposed drug during resection and an effective adjunct to radiation and TMZ.

Propofol Inhibits Glioma Stem Cell Growth and Migration and Their Interaction with Microglia via BDNF-AS and Extracellular Vesicles.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor. GBM contains a small subpopulation of glioma stem cells (GSCs) that are implicated in treatment resistance, tumor infiltration, and recurrence, and are thereby considered important therapeutic targets. Recent clinical studies have suggested that the choice of general anesthetic (GA), particularly propofol, during tumor resection, affects subsequent tumor response to treatments and patient prognosis. In this study, we investigated the molecular mechanisms underlying propofol's anti-tumor effects on GSCs and their interaction with microglia cells. Propofol exerted a dose-dependent inhibitory effect on the self-renewal, expression of mesenchymal markers, and migration of GSCs and sensitized them to both temozolomide (TMZ) and radiation. At higher concentrations, propofol induced a large degree of cell death, as demonstrated using microfluid chip technology. Propofol increased the expression of the lncRNA BDNF-AS, which acts as a tumor suppressor in GBM, and silencing of this lncRNA partially abrogated propofol's effects. Propofol also inhibited the pro-tumorigenic GSC-microglia crosstalk via extracellular vesicles (EVs) and delivery of BDNF-AS. In conclusion, propofol exerted anti-tumor effects on GSCs, sensitized these cells to radiation and TMZ, and inhibited their pro-tumorigenic interactions with microglia via transfer of BDNF-AS by EVs.

LncRNA GAS5 represses stemness and malignancy of gliomas via elevating the SPACA6-miR-125a/let-7e Axis.

Glioblastoma (GBM) is a highly invasive neurological malignancy with poor prognosis. LncRNA-GAS5 (growth arrest-specific transcript 5) is a tumor suppressor involved in multiple cancers. In this study, we explored the clinical significance, biological function, and underlying mechanisms of GAS5 in GBM. We showed that lncRNA-GAS5 expression decreased in high-grade glioma tissues and cells, which might be associated with poor prognosis. GAS5 overexpression lowered cell viability, suppressed GBM cell migration and invasion, and impaired the stemness and proliferation of glioma stem cells (GSCs). We further discovered that GAS5 inhibited the viability of glioma cells through miR-let-7e and miR-125a by protecting SPACA6 from degradation. Moreover, GAS5 played an anti-oncogenic role in GBM through the combined involvement of let-7e and miR-125a in vivo and in vitro. Notably, these two miRNAs block the IL-6/STAT3 pathway in tumor tissues extracted from a xenograft model. Taken together, our study provides evidence for an important role of GAS5 in GBM by affecting the proliferation and migration of GSCs, thus providing a new potential prognostic biomarker and treatment strategy for GBM.

CSIG-18. HYPERMETHYLATION OF SLIT-ROBO PATHWAY GENES RESULTS IN INACTIVATION IN GLIOMA PROGRESSION

Abstract INTRODUCTION Glioblastomas (GBM) are the most common and malignant primary brain tumors. Their rapid growth and invasion into neuronal parenchyma is devastating, with limited treatment options. Genomic alterations have been extensively studied in gliomas tumors, including aberrations of the Slit-Robo pathway genes. The Slit family of secreted proteins modulates migration of somatic cells during development and mediate their effect by binding to its receptor Roundabout (Robo). Genes in the Slit-Robo pathways have been shown to be inactivated by promoter hypermethylation in a number of human cancers. We hypothesize that Slit-Robo signaling pathways are modulated via promotor methylation, controlling GBM malignancy and regulating the invasive capacity of glioma stem cells (GSCs). METHODS We characterized expression of mRNA and protein via real-time PCR and Western blots in primary GBM tissue. We then assessed whether epigenetic alterations correlate with Slit-Robo expression by conducting in vitro demethylation assays in patient derived GSCs followed by real-time PCR. We conducted invasion assays to elucidate the role of Slit in GSC invasion. RESULTS The Cancer Genome Atlas indicates a negative correlation between Robo2 expression and glioma patient survival (17.1 months versus 37.4 months; p < 1.4E-4). We discovered differential expression of the Slit (1-3) and Robo (2, 3) genes and protein of up to 8-fold between grades 3 and 4 astrocytomas (8.34 versus 4.73 density). Treating patient derived glioma stem cells with 5-aza-2-deoxycytidine results in induction in Slit-Robo gene expression ranging from 5 – 40-fold (p < 0.01). Addition of Slit 2 and 3 in an invasion assay induced migration of GSCs in a differential and concentration dependent manner of 10 - 25% (p < 0.05). CONCLUSIONS Our results suggest that promoter methylation and gene expression of the Slit-Robo genes correlates with protein expression, tumor invasiveness, and prognosis and a potential therapeutic target.

Abstract 1153: Hypoxic microenvironments promote stemness and enhance the directed migration of glioma stem cells

Abstract Glioblastoma (GBM) is a highly malignant primary brain tumor and has a poor prognosis and is resistant to the most advanced therapies. The development of highly hypoxic centrally located necrotic regions is strongly associated with accelerated GBM progression, yet mechanistic explanations are not complete. Hypoxic regions within GBM are highly enriched for glioma stem cells (GSC), which are critical for tumor initiation, recurrence and therapeutic resistance due to their self-renewing capabilites. While most GBM cells migrate outward, away from hypoxia, GSCs accumulate there, forming a hypercellular border called pseudopalisades that promote their survival. Mechanisms that mediate GSC accumulation within pseudopalisades are unknown. We hypothesize that GSCs are enriched in the pseudopalisades due to the homing of a subpopulation towards the supportive hypoxic microenvironment. This in turn activates critical hypoxia-induced drivers to promote stemness, survival and migratory behavior encouraging the recruitment of more GSCs. Using two genetically distinct patient-derived GBM cell lines, we found that exposure to hypoxia in vitro led to enrichment of GSCs among a heterogeneous population of GSCs and non-GSCs. We found that hypoxia enhanced the expression of stem cell, pro-survival and pro-migratory factors in GSCs, such as the pro-migratory chemokine SDF-1α, and its cognate receptor CXCR4. Importantly, we found hypoxia also induced upregulation of OLIG2, one of four core transcription factors necessary and sufficient to induce stemness, in GSCs and non-GSCs exposed to hypoxia. We determined that OLIG2 was necessary for the expression of stem cell markers, and that its knockdown reduced cellular migration in hypoxia vs normoxia in GSCs. This suggests a critical role for OLIG2 in both hypoxia-induced stemness and homing behavior. We also generated a unique model of localized hypoxia in one brain hemisphere through vaso-cclusion and observed the homing of xenografted GBM cells towards hypoxia, regulated by OLIG2 in vivo. These data shed light on how hypoxic tumor microenvironments facilitate mechanisms vital to GBM progression and therapeutic resistance. Citation Format: Nitya V. Sharma, Monica Chau, Subhas Mukherjee, James Ross, Brandon Miller, Changming Zhang, Jun Kong, Carol Tucker-Burden, Emily Kaissi, John W. Reitnauer, Bilge Dundar, Cheryl L. Olson, Gregory Bix, Daniel J. Brat. Hypoxic microenvironments promote stemness and enhance the directed migration of glioma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1153.

Targeting Glioma Stem Cells by Functional Inhibition of Dynamin 2: A Novel Treatment Strategy for Glioblastoma

Glioma stem cells (GSCs) play major roles in drug resistance, tumour maintenance and recurrence of glioblastoma. We investigated inhibition of the GTPase dynamin 2 as a therapy for glioblastoma. Glioma cell lines and patient-derived GSCs were treated with dynamin inhibitors, Dynole 34-2 and CyDyn 4-36. We studied about cell viability, and GSC neurosphere formation in vitro and orthotopic tumour growth in vivo. Dynamin inhibition reduced glioblastoma cell line viability and suppressed neurosphere formation and migration of GSCs. Tumour growth was reduced by CyDyn 4-36 treatment. Dynamin 2 inhibition therefore represents a novel approach for stem cell-directed Glioblastoma therapy.

A three-dimensional (3D) organotypic microfluidic model for glioma stem cells – Vascular interactions

Glioblastoma (GBM) is one of the deadliest forms of cancer. Despite many treatment options, prognosis of GBM remains dismal with a 5-year survival rate of 4.7%. Even then, tumors often recur after treatment. Tumor recurrence is hypothesized to be driven by glioma stem cell (GSC) populations which are highly tumorigenic, invasive, and resistant to several forms of therapy. GSCs are often concentrated around the tumor vasculature, referred to as the vascular niche, which are known to provide microenvironmental cues to maintain GSC stemness, promote invasion, and resistance to therapies. In this work, we developed a 3D organotypic microfluidic platform, integrated with hydrogel-based biomaterials, to mimic the GSC vascular niche and study the influence of endothelial cells (ECs) on patient-derived GSC behavior and identify signaling cues that mediate their invasion and phenotype. The established microvascular network enhanced GSC migration within a 3D hydrogel, promoted invasive morphology as well as maintained GSC proliferation rates and phenotype (Nestin, SOX2, CD44). Notably, we compared migration behavior to in vivo mice model and found similar invasive morphology suggesting that our microfluidic system could represent a physiologically relevant in vivo microenvironment. Moreover, we confirmed that CXCL12-CXCR4 signaling is involved in promoting GSC invasion in a 3D vascular microenvironment by utilizing a CXCR4 antagonist (AMD3100), while also demonstrating the effectiveness of the microfluidic as a drug screening assay. Our model presents a potential ex vivo platform for studying the interplay of GSCs with its surrounding microenvironment as well as development of future therapeutic strategies tailored toward disrupting key molecular pathways involved in GSC regulatory mechanisms.

PO-291 A cell-penetrating peptide based on the connexin43-Src interacting sequence reduces glioma stem cell migration and proliferation by recruiting Src, Csk and PTEN

IntroductionThe expression of connexin43 (Cx43), the main gap junction channel-forming protein, is down-regulated in glioma stem cells (GSC). Our previous studies showed that a cell-penetrating peptide containing the region of Cx43 that interacts with the oncoprotein c-Src (TAT-Cx43266–283), inhibits its oncogenic activity and up-regulates the tumour suppressor PTEN. Through this pathway, TAT-Cx43266–283 reduces glioma cell proliferation and reverses the GSC phenotype. Our next goal was to uncover the mechanism of action and the effects of TAT-Cx43266–283 on the migration and invasion of human GSCs.Material and methodsHuman primary GSCs (G9, G12, G13, G15 and G16) were obtained from glioblastoma patients’ biopsies from the Neurosurgery Service of the Hospital Universitario de Salamanca, Spain. The migration was analysed in these primary GSCs by tracking individual cell trajectories in cultures recorded by Time-Lapse Live-cell Imaging and the invasion with Matrigel-treated transwell inserts. The same tumour biopsies were used to study the effect of TAT-Cx43266–283 on fresh undissociated tumour blocks by time-lapse microscopy. The molecular interactions were studied in G166 GSCs by pull-down assays of biotinylated TAT-Cx43266–283 (TAT-Cx43266–283-B) and Western blot analysis.Results and discussionsOur results confirmed that TAT-Cx43266–283 strongly reduces the migration and invasion of human primary GSCs by inhibiting c-Src activity and up-regulating PTEN that consequently, lowers the levels of phosphorylation of focal adhesion kinase tyrosines 397, 576 and 577. In addition, fresh undissociated tumour blocks revealed a dramatic reduction on the survival of these glioblastoma cells when exposed to TAT-Cx43266–283. By pull-down assays, we showed that TAT-Cx43266–283-B recruits c-Src together with its inhibitors PTEN and Csk, confirming the proposed mechanism for Cx43.ConclusionIn conclusion, TAT-Cx43266–283 exerts an important antitumor effect by inhibiting c-Src and up-regulating PTEN with the subsequent reduction in FAK phosphorylation required to establish appropriate focal adhesions for migration, proliferation and survival. This inhibition is mediated by the recruitment of the c-Src intrinsic inhibitors, PTEN and Csk. All together, these results reinforce the relevance of this sequence of Cx43 that interacts with c-Src for the development of new therapies against glioblastoma.

A Short Region of Connexin43 Reduces Human Glioma Stem Cell Migration, Invasion, and Survival through Src, PTEN, and FAK.

SummaryConnexin43 (CX43), a protein that forms gap junction channels and hemichannels in astrocytes, is downregulated in high-grade gliomas. Its relevance for glioma therapy has been thoroughly explored; however, its positive effects on proliferation are counterbalanced by its effects on migration and invasion. Here, we show that a cell-penetrating peptide based on CX43 (TAT-Cx43266-283) inhibited c-Src and focal adhesion kinase (FAK) and upregulated phosphatase and tensin homolog in glioma stem cells (GSCs) derived from patients. Consequently, TAT-Cx43266-283 reduced GSC motility, as analyzed by time-lapse microscopy, and strongly reduced their invasive ability. Interestingly, we investigated the effects of TAT-Cx43266-283 on freshly removed surgical specimens as undissociated glioblastoma blocks, which revealed a dramatic reduction in the growth, migration, and survival of these cells. In conclusion, a region of CX43 (amino acids 266–283) exerts an important anti-tumor effect in patient-derived glioblastoma models that includes impairment of GSC migration and invasion.

Abstract 5217: Microenvironmental influences on glioma stem cell migration

Abstract Glioblastoma (GBM) is the most aggressive of the primary brain tumors and carries a poor prognosis due to its highly infiltrative nature and tendency to progress. Glioma stem cells are a small tumor-initiating population in GBM whose properties are incompletely understood. Autocrine and paracrine factors associated with specific microenvironmental niches, such as the perivascular space and hypoxic regions, are thought to attract and support this stem cell component. Precise mechanisms guiding the homing to these niches have not been defined. Glioma stem cells express the receptor CXCR4, which initiates cell migration in the presence of the chemokine, stromal cell-derived factor-1α (SDF-1α). Both hypoxic and vascular regions in the brain express SDF-1α. Therefore, we hypothesize that glioma stem cells transplanted into the brain are driven by SDF-1α chemoattraction towards vascular and hypoxic niches. Cells were sorted for the stem cell marker CD133 using MACS for two explanted human GBM cell lines grown as neurospheres (N08-74 and N08-30). Sorted cells were compared to unsorted cells by Western blot for expression of CD133, hypoxia inducible factor-1α (HIF-1α), CXCR4 and stem cell markers under normoxia and hypoxia. Two million CD133-sorted cells suspended in PBS were delivered into the nasal mucosa of NOD/SCID mice to test cellular migration into the brain via intranasal transplantation. Coronal sections of brain tissue were analyzed at 12 hours after intranasal cell delivery. In both GBM cell lines (N08-30 and N08-74), we confirmed that sorted cells expressed more CD133 than unsorted cells and that both cell lines expressed CXCR4. Further, CXCR4 expression was significantly greater in the CD133-sorted N08-30 cells with a trend in an increase of c-Myc expression compared to unsorted cells. Hypoxic exposure of N08-74 CD133-sorted cells showed a trend of increased HIF-1α and c-Myc expression. In mice, we demonstrated that CD133-sorted cells can migrate into the brain when delivered intranasally. To show the potential for migration towards the vasculature, we show that SDF-1α is expressed in the brain vasculature in vivo. These data demonstrate that GBM cells can be sorted for CD133-positive glioma stem cells, respond to hypoxia, and express the SDF-1α receptor, CXCR4. In one cell line, CXCR4 expression increased after CD133 cell sorting. With intranasal delivery, CD133-sorted cells can migrate into the brain. Understanding the migration behavior of glioma stem cells relative to the diverse microenvironments has implications in treatment for targeting certain migratory populations or microenvironments. These data prepare the conditions to test the migratory potential of glioma stem cells in hypoxic and vascular environments in the brain. Citation Format: Monica J. Chau, Myles R. McCrary, Subhas Mukherjee, Daniel J. Brat. Microenvironmental influences on glioma stem cell migration. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5217. doi:10.1158/1538-7445.AM2015-5217

RTVP-1 promotes mesenchymal transformation of glioma via a STAT-3/IL-6-dependent positive feedback loop.

Glioblastomas (GBMs), the most aggressive primary brain tumors, exhibit increased invasiveness and resistance to anti-tumor treatments. We explored the role of RTVP-1, a glioma-associated protein that promotes glioma cell migration, in the mesenchymal transformation of GBM. Analysis of The Cancer Genome Atlas (TCGA) demonstrated that RTVP-1 expression was higher in mesenchymal GBM and predicted tumor recurrence and poor clinical outcome. ChiP analysis revealed that the RTVP-1 promoter binds STAT3 and C/EBPβ, two master transcription factors that regulate mesenchymal transformation of GBM. In addition, IL-6 induced RTVP-1 expression in a STAT3-dependent manner. RTVP-1 increased the migration and mesenchymal transformation of glioma cells. Similarly, overexpression of RTVP-1 in human neural stem cells induced mesenchymal differentiation, whereas silencing of RTVP-1 in glioma stem cells (GSCs) decreased the mesenchymal transformation and stemness of these cells. Silencing of RTVP-1 also increased the survival of mice bearing GSC-derived xenografts. Using gene array analysis of RTVP-1 silenced glioma cells we identified IL-6 as a mediator of RTVP-1 effects on the mesenchymal transformation and migration of GSCs, therefore acting in a positive feedback loop by upregulating RTVP-1 expression via the STAT3 pathway. Collectively, these results implicate RTVP-1 as a novel prognostic marker and therapeutic target in GBM.

CS-09 * RTVP-1 PROMOTES THE MESENCHYMAL TRANSFORMATION OF GLIOMA STEM CELLS VIA THE CXCR4 AND IL-6 PATHWAYS

Glioblastoma (GBM), the most aggressive primary brain tumors, are categorized into the major subgroups: proneural, neural, classical and mesenchymal, the latter being characterized by increased invasion and poor prognosis. We recently identified RTVP-1 as a glioma-associated protein that regulates glioma cell migration and invasion. Using ChiP analyses, we found that the RTVP-1 promoter binds STAT3 and C/EBPbeta, the two master transcription factors that regulate mesenchymal transformation of GBM. Analysis of TCGA tumor specimens demonstrated that the expression of RTVP-1 was higher in the mesenchymal GBM and was inversely correlated with patient survival. We further found that RTVP-1 was expressed in glioma stem cells (GSCs) but not in human neural stem cells (NSCs). Overexpression of RTVP-1 in NSCs induced their mesenchymal transformation, whereas silencing of RTVP-1 in GSCs decreased their mesenchymal signature, increased their neural phenotypes and inhibited the self renewal and stemness of these cells. Silencing of RTVP-1 also decreased tumor volume of GSC-derived xenografts and increased animal survival. Using gene array analysis of RTVP-1 silenced cells we identified IL-6 and CXCR4 as major mediators of RTVP-1 effects on the mesenchymal transformation and self-renewal of GSCs. Using a pull down assay with His-tagged RTVP-1 and FRET analysis, we identified HSP27, N-WASP and hnRNPK as novel interacting proteins of RTVP-1, that mediate its effects on GSC migration and invadopodia formation. In summary, RTVP-1 expression is regulated by STAT3 and CEBPbeta and is promoting the mesenchymal transformation of GSCs. RTVP-1 induces self-renewal and migration of GSCs by the increased expression of IL-6 and CXCR4 and via its interaction with N-WASP, hnRNPK and HSP27. The upregulation of IL-6 by RTVP-1 acts in a positive feedback loop to further increase RTVP-1 expression by activating the STAT3 pathway. Collectively, these results implicate RTVP-1 as a novel prognostic marker and therapeutic target in GBM.

ET-30 * A PHASE I TRIAL OF AZD7451, A TROPOMYOSIN-RECEPTOR KINASE (TRK) INHIBITOR, FOR ADULTS WITH RECURRENT GLIOBLASTOMA MULTIFORME (GBM)

BACKGROUND: We have shown that inhibition of TrkA both genetically and pharmacologically results in profound inhibition of glioma stem cell migration in vitro and in vivo. AZD7451 is the first in class of highly potent and selective Trk inhibitors and therefore may have significant anti-glioma cell invasive properties. METHODS: We conducted an open label phase I study of AZD 7451 mesylate, with a standard 3 + 3 dose escalation scheme, to determine the MTD and describe pharmacokinetics. The drug was administered as an oral solution (10 mg/ml) at 12 mg, 24 mg, 40 mg, and 60 mg daily. DL3 and DL4 were administered with divided twice daily dosing. Plasma concentrations of AZD7451 were measured with ultra-performance liquid chromatography and tandem mass spectrometry using samples obtained at baseline and various points through the first 4 week cycle of therapy. RESULTS: 14 patients were enrolled. There were no radiographic responses to therapy, and no patients achieved disease control beyond cycle 2. There were no DLTs. Unexpected treatment related toxicities included grade 1-2 thrombocytopenia and anemia. No neurological adverse events related to therapy were observed. All patients were evaluable for C1D1 pharmacokinetics. The majority of samples were below the limit of quantitation (LLOQ) of 0.5 ng/mL by ∼ 4 h post-dose. AZD7451 T1/2 ranged from 20-50 min with once daily or BID dosing. No statistically significant increases in CMAX, AUCINF, T1/2, or volume of distribution were observed as dose increased. CONCLUSIONS: Based on the interim PK analysis for AZD7451 indicating poor oral bioavailability, short <60 min plasma T1/2 half-life, and rapid clearance kinetics, PK exposures are insufficient in humans to inhibit Trk at target plasma concentrations necessary for efficacy in preclinical models. The absence of DLT's or early efficacy signals in GBM patients is also consistent with poor AZD7451 exposures in humans.

The role of Src family kinases in growth and migration of glioma stem cells

Src family kinases (SFKs) are highly expressed and active in clinical glioblastoma multiforme (GBM) specimens. SFKs inhibitors have been demonstrated to inhibit proliferation and migration of glioma cells. However, the role of SFKs in glioma stem cells (GSCs), which are important for treatment resistance and recurrence, has not been reported. Here, we examined the expression pattern of individual members of SFKs and their functional role in CD133+ GSCs in comparison to primary glioma cells. We found that Fyn, c-Src and Yes were robustly expressed in GSCs while Lck was absent. Knockdown of c-Src, Yes or treatment with the SFK inhibitor dasatinib inhibited the migration of GSCs, but had no impact on their growth or self-renewal. These results suggest that SFKs represent an effective target for GSC migration but not for their growth.

Effect of RTVP-1 on the mesenchymal transformation of glioma stem cells (GSCs) and their self-renewal and migration.

2073 Background: Glioblastoma (GBM), the most aggressive primary brain tumors, exhibit increased invasiveness and resistance to anti-tumor treatments. GBM are categorized into proneural, neural, classical and mesenchymal subgroups, the latter being characterized by increased invasion and poor prognosis. We recently reported that RTVP-1 is highly expressed in GBM and its expression is correlated with astrocytic tumor grade. Methods: We employed promoter and Chip analyses, analysis of tumor specimens submitted to TCGA , GSC self-renewal and migration assays, mesenchymal and neural differentiation, gene array analysis and pull-down assay followed by FRET. Results: The RTVP-1 promoter binds STAT3 and C/EBPb, the transcription factors that regulate mesenchymal transformation of GBM. The expression of RTVP-1 is higher in mesenchymal GBM and is inversely correlated with patient survival in the proneural group. We examined the expression and functions of RTVP-1 in GSCs, a small population of cancer stem cells that are implicated in the increased migration, radio- and chemo-resistance of GSCs and tumor recurrence. RTVP-1 was expressed in the different GSCs but not in the normal human neural stem cells (NSCs). Overexpression of RTVP-1 in NSCs induced their mesenchymal transformation, whereas silencing of RTVP-1 in GSCs decreased their mesenchymal and increased their neural phenotypes. Moreover, RTVP-1 promoted the self-renewal and migration of GSCs. Using gene array analysis of RTVP-1 silenced cells we identified IL-6 and CXCR4 as major mediators of RTVP-1 effects on the mesenchymal transformation and self-renewal of GSCs. In addition, using a pull down assay with His-tagged RTVP-1 we identified N-WASP and hnRNPK as novel interacting proteins of RTVP-1 that mediate its effects on glioma cell migration. Conclusions: RTVP-1 expression is associated with mesenchymal transformation of GSCs. RTVP-1 promotes self-renewal and migration of GSCs and these effects are mediated by the increased expression of IL-6 and CXCR4 and via interaction with N-WASP and hnRNPK. Collectively, these results suggest that RTVP-1 may represent a novel diagnostic marker and a therapeutic target in GBM.

Abstract 5288: Glioma stem cells stimulate the motility of brain endothelial cells: Identification of cell-adhesion molecules mediating motility and direct interaction

Abstract Angiogenesis is a prominent characteristic of malignant glioma. Glioma stem cells (GSCs) reside adjacent to blood vessels in the perivascular niche, and recent evidence suggests a role for GSCs in promoting angiogenesis. Here we examined the interplay between GSCs and endothelial cells (ECs) on a laminin substrate, and the potential for GSC promotion of EC motility. In an in vitro 2D motility assay, we found that GSCs dramatically stimulated EC migration, and ECs also stimulated GSC migration. An antibody to integrin α6 blocked in part the motility of both GSCs and ECs. We also found that GSCs directly interact with ECs; therefore, we investigated which cell-adhesion molecules mediated the interaction. In a cell-cell binding assay, blocking antibodies to both L1-cell adhesion molecule (L1CAM) and integrin αvβ3 inhibited in part the direct contact between GSCs and ECs. In the motility assay, the combination of antibodies to L1CAM and αvβ3 significantly inhibited the motility and interaction of both cell types. These data suggest GSCs can promote angiogenesis by promoting EC migration, and indicate a role for the cell-adhesion receptors L1CAM and αvβ3 in the direct interaction between GSCs and ECs that may promote EC motility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5288. doi:1538-7445.AM2012-5288