Neuronal differentiation drives the antitumor activity of mitogen-activated protein kinase kinase (MEK) inhibition in glioblastoma.

Background: Epidermal growth factor receptor (EGFR) amplification is found in nearly 40%-50% of glioblastoma cases. Several EGFR inhibitors have been tested in glioblastoma but have failed to demonstrate long-term therapeutic benefit, presumably because of acquired resistance. Targeting EGFR downstream signaling with mitogen-activated protein kinase kinase 1 and 2 (MEK1/2) inhibitors would be a more effective approach to glioblastoma treatment. We tested the therapeutic potential of MEK1/2 inhibitors in glioblastoma using 3D cultures of glioma stem-like cells (GSCs) and mouse models of glioblastoma. Methods: Several MEK inhibitors were screened in an unbiased high-throughput platform using GSCs. Cell death was evaluated using flow cytometry and Western blotting (WB) analysis. RNA-seq, real-time quantitative polymerase chain reaction, immunofluorescence, and WB analysis were used to identify and validate neuronal differentiation. Results: Unbiased screening of multiple MEK inhibitors in GSCs showed antiproliferative and apoptotic cell death in sensitive cell lines. An RNA-seq analysis of cells treated with trametinib, a potent MEK inhibitor, revealed upregulation of neurogenesis and neuronal differentiation genes, such as achaete-scute homolog 1 (ASCL1), delta-like 3 (DLL3), and neurogenic differentiation 4 (NeuroD4). We validated the neuronal differentiation phenotypes in vitro and in vivo using selected differentiation markers (ß-III-tubulin, ASCL1, DLL3, and NeuroD4). Oral treatment with trametinib in an orthotopic GSC xenograft model significantly improved animal survival, with 25%-30% of mice being long-term survivors. Conclusions: Our findings demonstrated that MEK1/2 inhibition promotes neuronal differentiation in glioblastoma, a potential additional mechanism of action of MEK1/2 inhibitors. Thus, MEK inhibitors could be efficacious in glioblastoma patients with activated EGFR/MAPK signaling.

Depletion of CLK2 sensitizes glioma stem-like cells to PI3K/mTOR and FGFR inhibitors.

The Cdc2-like kinases (CLKs) regulate RNA splicing and have been shown to suppress cell growth. Knockdown of CLK2 was found to block glioma stem-like cell (GSC) growth in vivo through the AKT/FOXO3a/p27 pathway without activating mTOR and MAPK signaling, suggesting that these pathways mediate resistance to CLK2 inhibition. We identified CLK2 binding partners using immunoprecipitation assays and confirmed their interactions in vitro in GSCs. We then tested the cellular viability of several signaling inhibitors in parental and CLK2 knockdown GSCs. Our results demonstrate that CLK2 binds to 14-3-3τ isoform and prevents its ubiquitination in GSCs. Stable CLK2 knockdown increased PP2A activity and activated PI3K signaling. Treatment with a PI3K/mTOR inhibitor in CLK2 knockdown cells led to a modest reduction in cell viability compared to drug treatment alone at a lower dose. However, FGFR inhibitor in CLK2 knockdown cells led to a decrease in cell viability and increased apoptosis. Reduced expression of CLK2 in glioblastoma, in combination with FGFR inhibitors, led to synergistic apoptosis induction and cell cycle arrest compared to blockade or either kinase alone.

The polo-like kinase 1 inhibitor volasertib synergistically increases radiation efficacy in glioma stem cells.

BACKGROUND: Despite the availability of hundreds of cancer drugs, there is insufficient data on the efficacy of these drugs on the extremely heterogeneous tumor cell populations of glioblastoma (GBM). RESULTS: The PKIS of 357 compounds was initially evaluated in 15 different GSC lines which then led to a more focused screening of the 21 most highly active compounds in 11 unique GSC lines using HTS screening for cell viability. We further validated the HTS result with the second-generation PLK1 inhibitor volasertib as a single agent and in combination with ionizing radiation (IR). In vitro studies showed that volasertib inhibited cell viability, and high levels of the anti-apoptotic protein Bcl-xL expression were highly correlated with volasertib resistance. Volasertib sensitized GSCs to radiation therapy by enhancing G2/M arrest and by inducing apoptosis. Colony-formation assay demonstrated that volasertib plus IR synergistically inhibited colony formation. In intracranial xenograft mouse models, the combination of volasertib and radiation significantly inhibited GSC tumor growth and prolonged median survival compared with radiation treatment alone due to inhibition of cell proliferation, enhancement of DNA damage, and induction of apoptosis. CONCLUSIONS: Our results reinforce the potential therapeutic efficacy of volasertib in combination with radiation for the treatment of GBM. METHODS: We used high-throughput screening (HTS) to identify drugs, out of 357 compounds in the published Protein Kinase Inhibitor Set, with the greatest efficacy against a panel of glioma stem cells (GSCs), which are representative of the classic cancer genome atlas (TCGA) molecular subtypes.

Targeting intercellular adhesion molecule-1 prolongs survival in mice bearing bevacizumab-resistant glioblastoma.

Intercellular cell adhesion molecule 1 (ICAM-1; also known as CD54) is overexpressed in bevacizumab-resistant glioblastoma. In the present study, we tested our hypothesis that highly expressed ICAM-1 mediates glioblastoma's resistance to antiangiogenic therapy. We validated ICAM-1 overexpression in tumors resistant to antiangiogenic therapy using real-time polymerase chain reaction, immunohistochemistry, and Western blotting. We also detected ICAM1 expression in most glioma stem cells (GSCs). We investigated the mechanism of ICAM-1 overexpression after bevacizumab treatment and found that ICAM-1 protein expression was markedly increased in a time-dependent manner in GSC11 and GSC17 cells under hypoxic conditions in vitro. We also found that hypoxia induced ICAM-1 overexpression through the up-regulation of phosphorylated signal transducer and activator of transcription (p-STAT3). Hypoxia-induced p-STAT3 increased the mRNA transcription of ICAM-1, which we could inhibit with the STAT3 inhibitor AZD1480. Next, we used GFP-tagged ICAM-1 shRNA lentivirus to knock down ICAM-1 in GSC11 and GSC17 glioma cell lines. Then, we injected shICAM-1 GSC11 and scramble glioma stem cells into the brains of nude mice. Mice bearing tumors from shICAM-1 GSC11 cells survived significantly longer than mice injected with control cells did. The tumor sizes was significantly decreased in mice bearing tumors from shICAM-1 cells than that in mice bearing tumors from GFP-tagged GSC11 control cells. Knocking down ICAM-1 suppressed tumor invasion in vitro and in vivo and inhibited macrophage infiltration to the tumor site in bevacizumab-treated mice. Our findings suggest that ICAM-1 is a potentially important mediator of tumor migration and invasion in bevacizumab-resistant glioblastoma. Targeting ICAM-1 may provide a new strategy for enhancing the efficacy of antiangiogenic therapy against glioblastoma and preventing the invasive phenotype of the disease.

Stabilization of phosphofructokinase 1 platelet isoform by AKT promotes tumorigenesis.

Phosphofructokinase 1 (PFK1) plays a critical role in glycolysis; however, its role and regulation in tumorigenesis are not well understood. Here, we demonstrate that PFK1 platelet isoform (PFKP) is the predominant PFK1 isoform in human glioblastoma cells and its expression correlates with total PFK activity. We show that PFKP is overexpressed in human glioblastoma specimens due to an increased stability, which is induced by AKT activation resulting from phosphatase and tensin homologue (PTEN) loss and EGFR-dependent PI3K activation. AKT binds to and phosphorylates PFKP at S386, and this phosphorylation inhibits the binding of TRIM21 E3 ligase to PFKP and the subsequent TRIM21-mediated polyubiquitylation and degradation of PFKP. PFKP S386 phosphorylation increases PFKP expression and promotes aerobic glycolysis, cell proliferation, and brain tumor growth. In addition, S386 phosphorylation in human glioblastoma specimens positively correlates with PFKP expression, AKT S473 phosphorylation, and poor prognosis. These findings underscore the potential role and regulation of PFKP in human glioblastoma development.Phosphofructokinase 1 (PFK1) plays a critical role in glycolysis. Here the authors show that PFK1 platelet isoform is upregulated in Glioblastoma and is required for tumor growth mechanistically, such upregulation is due to an increased stability induced by AKT activation via phosphorylation on residue S386.

Periostin (POSTN) Regulates Tumor Resistance to Antiangiogenic Therapy in Glioma Models.

Periostin (POSTN) interacts with multiple integrins to coordinate a variety of cellular processes, including epithelial-to-mesenchymal transition (EMT) and cell migration. In our previous study, anti-VEGF-A therapy was associated with resistance and EMT. This study sought to determine the role of POSTN in the resistance of glioma stem cells (GSC) to antiangiogenic therapy. In mouse xenograft models of human glioma, POSTN expression was associated with acquired resistance to anti-VEGF-A therapy and had a synergistic effect with bevacizumab in prolonging survival and decreasing tumor volume. Resistance to anti-VEGF-A therapy regulated by POSTN was associated with increased expression of TGFß1 and hypoxia-inducible factor-1α (HIF1α) in GSCs. At the molecular level, POSTN regulated invasion and expression of EMT (caveolin-1) and angiogenesis-related genes (HIF1α and VEGF-A) through activation of STAT3. Moreover, recombinant POSTN increased GSC invasion. Collectively, our findings suggest that POSTN plays an important role in glioma invasion and resistance to antiangiogenic therapy. Mol Cancer Ther; 15(9); 2187-97. ©2016 AACR.

Cdc2-like kinase 2 is a key regulator of the cell cycle via FOXO3a/p27 in glioblastoma.

Cdc2-like kinase 2 (CLK2) is known as a regulator of RNA splicing that ultimately controls multiple physiological processes. However, the function of CLK2 in glioblastoma progression has not been described. Reverse-phase protein array (RPPA) was performed to identify proteins differentially expressed in CLK2 knockdown cells compared to controls. The RPPA results indicated that CLK2 knockdown influenced the expression of survival-, proliferation-, and cell cycle-related proteins in GSCs. Thus, knockdown of CLK2 expression arrested the cell cycle at the G1 and S checkpoints in multiple GSC lines. Depletion of CLK2 regulated the dephosphorylation of AKT and decreased phosphorylation of Forkhead box O3a (FOXO3a), which not only translocated to the nucleus but also increased p27 expression. In two glioblastoma xenograft models, the survival duration of mice with CLK2-knockdown GSCs was significantly longer than mice with control tumors. Additionally, tumor volumes were significantly smaller in CLK2-knockdown mice than in controls. Knockdown of CLK2 expression reduced the phosphorylation of FOXO3a and decreased Ki-67 in vivo. Finally, high expression of CLK2 protien was significantly associated with worse patient survival. These findings suggest that CLK2 plays a critical role in controlling the cell cycle and survival of glioblastoma via FOXO3a/p27.

Novel MET/TIE2/VEGFR2 inhibitor altiratinib inhibits tumor growth and invasiveness in bevacizumab-resistant glioblastoma mouse models.

BACKGROUND: Glioblastoma highly expresses the proto-oncogene MET in the setting of resistance to bevacizumab. MET engagement by hepatocyte growth factor (HGF) results in receptor dimerization and autophosphorylation mediating tumor growth, invasion, and metastasis. Evasive revascularization and the recruitment of TIE2-expressing macrophages (TEMs) are also triggered by anti-VEGF therapy. METHODS: We investigated the activity of altiratinib (a novel balanced inhibitor of MET/TIE2/VEGFR2) against human glioblastoma stem cell lines in vitro and in vivo using xenograft mouse models. The biological activity of altiratinib was assessed in vitro by testing the expression of HGF-stimulated MET phosphorylation as well as cell viability after altiratinib treatment. Tumor volume, stem cell and mesenchymal marker levels, microvessel density, and TIE2-expressing monocyte infiltration were evaluated in vivo following treatment with a control, bevacizumab alone, bevacizumab combined with altiratinib, or altiratinib alone. RESULTS: In vitro, HGF-stimulated MET phosphorylation was completely suppressed by altiratinib in GSC17 and GSC267, and altiratinib markedly inhibited cell viability in several glioblastoma stem cell lines. More importantly, in multiple xenograft mouse models, altiratinib combined with bevacizumab dramatically reduced tumor volume, invasiveness, mesenchymal marker expression, microvessel density, and TIE2-expressing monocyte infiltration compared with bevacizumab alone. Furthermore, in the GSC17 xenograft model, altiratinib combined with bevacizumab significantly prolonged survival compared with bevacizumab alone. CONCLUSIONS: Together, these data suggest that altiratinib may suppress tumor growth, invasiveness, angiogenesis, and myeloid cell infiltration in glioblastoma. Thus, altiratinib administered alone or in combination with bevacizumab may overcome resistance to bevacizumab and prolong survival in patients with glioblastoma.

Interferon-regulatory factor-1 (IRF1) regulates bevacizumab induced autophagy.

PURPOSE: Antiangiogenic therapy is commonly being used for the treatment of glioblastoma. However, the benefits of angiogenesis inhibitors are typically transient and resistance often develops. Determining the mechanism of treatment failure of the VEGF monoclonal antibody bevacizumab for malignant glioma would provide insight into approaches to overcome therapeutic resistance. EXPERIMENTAL DESIGN: In this study, we evaluated the effects of bevacizumab on the autophagy of glioma cells and determined target genes involving in the regulation of bevacizumab-induced autophagy. RESULTS: We demonstrated that bevacizumab treatment increased expression of autophagy markers and autophagosome formation in cell culture experiments as well as in in vivo studies. Gene expression profile analysis performed on murine xenograft models of glioblastoma showed increased transcriptional levels of STAT1/IRF1 signaling in bevacizumab resistant tumors compared to control tumors. In vitro experiments showed that bevacizumab treatment increased IRF1 expression in a dose and time dependent manner, which was coincident with bevacizumab-mediated autophagy. Down regulation of IRF1 by shRNA blocked autophagy and increased AIF-dependent apoptosis in bevacizumab-treated glioma cells. Consistently, IRF1 depletion increased the efficacy of anti-VEGF therapy in a glioma xenograft model, which was due to less bevacizumab-promoted autophagy and increased apoptosis in tumors with down-regulated IRF1. CONCLUSIONS: These data suggest that IRF1 may regulate bevacizumab-induced autophagy, and may be one important mediator of glioblastoma resistant to bevacizumab.

Neutrophils promote the malignant glioma phenotype through S100A4.

PURPOSE: Antiangiogenic therapy is effective in blocking vascular permeability, inhibiting vascular proliferation, and slowing tumor growth, but studies in multiple cancer types have shown that tumors eventually acquire resistance to blockade of blood vessel growth. Currently, the mechanisms by which this resistance occurs are not well understood. EXPERIMENTAL DESIGN: In this study, we evaluated the effects of neutrophils on glioma biology both in vitro and in vivo and determined target genes by which neutrophils promote the malignant glioma phenotype during anti-VEGF therapy. RESULTS: We found that an increase in neutrophil infiltration into tumors is significantly correlated with glioma grade and in glioblastoma with acquired resistance to anti-VEGF therapy. Our data demonstrate that neutrophils and their condition media increased the proliferation rate of glioblastoma-initiating cells (GIC). In addition, neutrophils significantly increased GICs Transwell migration compared with controls. Consistent with this behavior, coculture with neutrophils promoted GICs to adopt morphologic and gene expression changes consistent with a mesenchymal signature. Neutrophil-promoting tumor progression could be blocked by S100A4 downregulation in vitro and in vivo. Furthermore, S100A4 depletion increased the effectiveness of anti-VEGF therapy in glioma. CONCLUSIONS: Collectively, these data suggest that increased recruitment of neutrophils during anti-VEGF therapy promotes glioma progression and may promote treatment resistance. Tumor progression with mesenchymal characteristics is partly mediated by S100A4, the expression of which is increased by neutrophil infiltration. Targeting granulocytes and S100A4 may be effective approaches to inhibit the glioma malignant phenotype and diminish antiangiogenic therapy resistance.

Acquired resistance to anti-VEGF therapy in glioblastoma is associated with a mesenchymal transition.

PURPOSE: Antiangiogenic therapy reduces vascular permeability and delays progression but may ultimately promote an aggressive treatment-resistant phenotype. The aim of the present study was to identify mechanisms responsible for glioblastoma resistance to antiangiogenic therapy. EXPERIMENTAL DESIGN: Glioma stem cell (GSC) NSC11 and U87 cell lines with acquired resistance to bevacizumab were developed from orthotopic xenografts in nude mice treated with bevacizumab. Genome-wide analyses were used to identify changes in tumor subtype and specific factors associated with resistance. RESULTS: Mice with established parental NSC11 and U87 cells responded to bevacizumab, whereas glioma cell lines derived at the time of acquired resistance to anti-VEGF therapy were resistant to bevacizumab and did not have prolongation of survival compared with untreated controls. Gene expression profiling comparing anti-VEGF therapy-resistant cell lines to untreated controls showed an increase in genes associated with a mesenchymal origin, cellular migration/invasion, and inflammation. Gene-set enrichment analysis showed that bevacizumab-treated tumors showed a highly significant correlation to published mesenchymal gene signatures. Mice bearing resistant tumors showed significantly greater infiltration of myeloid cells in NSC11- and U87-resistant tumors. Invasion-related genes were also upregulated in both NSC11 and U87 resistant cells which had higher invasion rates in vitro compared with their respective parental cell lines. CONCLUSIONS: Our studies identify multiple proinflammatory factors associated with resistance and identify a proneural to mesenchymal transition in tumors resistant to antiangiogenic therapy.

Cytokines associated with toxicity in the treatment of recurrent glioblastoma with aflibercept.

Plasma profiling of patients treated with antiangiogenic agents may identify markers that correlate with toxicity. Objectives were to correlate changes in cytokine and angiogenic factors as potential markers of toxicity to aflibercept. Circulating cytokine and angiogenic factors were measured in 28 patients with recurrent glioblastoma in a single-arm phase II study of aflibercept. Plasma samples were analyzed at baseline, 24 h, and 28 days using multiplex assays or ELISA. We evaluated log-transformed baseline biomarker expressions with Cox proportional hazard regression models to assess the effect of markers on any grade II-IV (Gr II-IV) toxicity, on-target toxicity (hypertension, proteinuria, thromboembolism), and fatigue. All tests were two sided with a statistical significance level of p = 0.05. Among 28 pts, there were 116 Gr II-IV events. Changes in IL-13 from baseline to 24 h predicted on-target toxicities. Increases in IL-1b, IL-6, and IL-10 at 24 h were significantly associated with fatigue. Progression-free survival was 14.9 months for patients in the all-toxicity group and 9.0 months for patients in the on-target toxicity group compared to 4.3 months for those who did not develop any Gr II-IV toxicity (p = 0.002 and p = 0.045, respectively). Toxicity from antiangiogenic therapy remains an important cause of antiangiogenic treatment discontinuation and patient morbidity. Changes in IL6, IL10, and IL13 were repeatedly correlated with toxicity. Profiling of IL-13 as a surrogate for endothelial dysfunction could individualize patients at risk during antiangiogenic therapy, as could identifying those at higher risk for fatigue using IL-6 and IL-10.

Modulating antiangiogenic resistance by inhibiting the signal transducer and activator of transcription 3 pathway in glioblastoma.

Determining the mechanism of treatment failure of VEGF signaling inhibitors for malignant glioma patients would provide insight into approaches to overcome therapeutic resistance. In this study, we demonstrate that human glioblastoma tumors failing bevacizumab have an increase in the mean percentage of p-STAT3-expressing cells compared to samples taken from patients failing non-antiangiogenic therapy containing regimens. Likewise, in murine xenograft models of glioblastoma, the mean percentage of p-STAT3-expressing cells in the gliomas resistant to antiangiogenic therapy was markedly elevated relative to controls. Administration of the JAK/STAT3 inhibitor AZD1480 alone and in combination with cediranib reduced tumor hypoxia and the infiltration of VEGF inhibitor-induced p-STAT3 macrophages. Thus, the combination of AZD1480 with cediranib markedly reduced tumor volume, and microvascular density, indicating that up regulation of the STAT3 pathway can mediate resistance to antiangiogenic therapy and combinational approaches may delay or overcome resistance.

Glioblastoma resistance to anti-VEGF therapy is associated with myeloid cell infiltration, stem cell accumulation, and a mesenchymal phenotype.

Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis. Inhibiting the VEGF-VEGF receptor (R) signal transduction pathway in glioblastoma has recently been shown to delay progression, but the relative benefit and mechanisms of response and failure of anti-VEGF therapy and VEGFR inhibitors are not well understood. The purpose of our study was to evaluate the relative effectiveness of VEGF sequestration and/or VEGFR inhibition on orthotopic tumor growth and the mechanism(s) of treatment resistance. We evaluated, not only, the effects of anti-VEGF therapy (bevacizumab), anti-VEGFR therapy (sunitinib), and the combination on the survival of mice bearing orthotopic gliomas, but also the differential effects of the treatments on tumor vascularity, cellular proliferation, mesenchymal and stem cell markers, and myeloid cell infiltration using flow cytometry and immunohistochemistry. Bevacizumab significantly prolonged survival compared with the control or sunitinib alone. Both antiangiogenic agents initially reduced infiltration of macrophages and tumor vascularity. However, multitargeted VEGFR inhibition, but not VEGF sequestration, rapidly created a vascular gradient and more rapidly induced tumor hypoxia. Re-infiltration of macrophages was associated with the induction of hypoxia. Combination treatment with bevacizumab and sunitinib improved animal survival compared with bevacizumab therapy alone. However, at the time of tumor progression, a significant increase in CD11b(+)/Gr1(+) granulocyte infiltration was observed, and tumors developed aggressive mesenchymal features and increased stem cell marker expression. Collectively, our results demonstrate a more prolonged decrease in tumor vascularity with bevacizumab than with sunitinib, associated with a delay in the development of hypoxia and sustained reduction of infiltrated myeloid cells.

Myeloid biomarkers associated with glioblastoma response to anti-VEGF therapy with aflibercept.

PURPOSE: VEGF and infiltrating myeloid cells are known regulators of tumor angiogenesis and vascular permeability in glioblastoma. We investigated potential blood-based markers associated with radiographic changes to aflibercept, which binds VEGF and placental growth factor (PlGF) in patients with recurrent glioblastoma. EXPERIMENTAL DESIGN: In this single-arm phase II trial, aflibercept was given intravenously every two weeks until disease progression. Plasma and peripheral blood mononuclear cells were collected at baseline and 24 hours, 14 days, and 28 days posttreatment. Plasma cytokines and angiogenic factors were quantified by using ELISA and multiplex bead assays, and myeloid cells were assessed by flow cytometry in a subset of patients. RESULTS: Circulating levels of VEGF significantly decreased 24 hours after treatment with aflibercept, coincident with radiographic response observed by MRI. PlGF initially decreased 24 hours posttreatment but increased significantly by days 14 and 28. Lower baseline levels of PlGF, elevated baseline levels of CTACK/CCL27, MCP3/CCL7, MIF, and IP-10/CXCL10, and a decrease in VEGFR1(+) monocytes from baseline to 24 hours were all associated with improved response. Tumor progression was associated with increases in circulating matrix metalloproteinase 9. CONCLUSIONS: These data suggest that decreases in VEGF posttreatment are associated with radiographic response to aflibercept. Elevated baseline chemokines of monocyte lineage in responding patients supports a role for myeloid cells and chemokines as potential biomarkers and regulators of glioma angiogenesis.

Tumor invasion after treatment of glioblastoma with bevacizumab: radiographic and pathologic correlation in humans and mice.

Patients with recurrent malignant glioma treated with bevacizumab, a monoclonal antibody to vascular endothelial growth factor (VEGF), alone or in combination with irinotecan have had impressive reductions in MRI contrast enhancement and vasogenic edema. Responses to this regimen, as defined by a decrease in contrast enhancement, have led to significant improvements in progression-free survival rates but not in overall survival duration. Some patients for whom this treatment regimen fails have an uncharacteristic pattern of tumor progression, which can be observed radiographically as an increase in hyperintensity on T2-weighted or fluid-attenuated inverse recovery (FLAIR) MRI. To date, there have been no reports of paired correlations between radiographic results and histopathologic findings describing the features of this aggressive tumor phenotype. In this study, we correlate such findings for 3 illustrative cases of gliomas that demonstrated an apparent phenotypic shift to a predominantly infiltrative pattern of tumor progression after treatment with bevacizumab. Pathologic examination of abnormal FLAIR areas on MRI revealed infiltrative tumor with areas of thin-walled blood vessels, suggesting vascular "normalization," which was uncharacteristically adjacent to regions of necrosis. High levels of insulin-like growth factor binding protein-2 and matrix metalloprotease-2 expression were seen within the infiltrating tumor. In an attempt to better understand this infiltrative phenotype associated with anti-VEGF therapy, we forced a highly angiogenic, noninvasive orthotopic U87 xenograft tumor to become infiltrative by treating the mice with bevacizumab. This model mimicked many of the histopathologic findings from the human cases and will augment the discovery of alternative or additive therapies to prevent this type of tumor recurrence in clinical practice.

Mediators of glioblastoma resistance and invasion during antivascular endothelial growth factor therapy.

PURPOSE: Vascular endothelial growth factor (VEGF) has been identified as a critical regulator of angiogenesis. Currently, several different strategies are being used to target the VEGF-VEGF receptor signal transduction pathway in glioblastoma. Although anti-VEGF therapy seems be effective in normalizing abnormal tumor vasculature, leading to an enhanced response to radiation and chemotherapy, tumors eventually become resistant to the therapy and adopt a highly infiltrative and invasive phenotype. EXPERIMENTAL DESIGN: In the present study, we evaluated the effects of anti-VEGF therapy (bevacizumab) on glioblastoma invasion both in vitro and in vivo and evaluated the angiogenesis- and invasion-related mediators of developed resistance to this therapy. RESULTS: We found that glioblastoma tumors escaped from antiangiogenic treatment by (a) reactivating angiogenesis through up-regulation of other proangiogenic factors and (b) invading normal brain areas, which was seen in association with up-regulation of matrix metalloproteinase (MMP)-2, MMP-9, and MMP-12; secreted protein, acidic, cysteine-rich; and tissue inhibitor of metalloproteinase 1. In addition to the paracrine effects of VEGF on endothelial cells, autocrine VEGF signaling seemed to regulate glioblastoma invasion because anti-VEGF therapy increased tumor invasiveness in vitro. CONCLUSIONS: Collectively, these findings reinforce the importance of VEGF in regulating tumor invasion and identify potential mediators of resistance to targeted VEGF therapy. These results will be important for developing novel combination therapies to overcome this resistance phenotype.

Dasatinib-induced autophagy is enhanced in combination with temozolomide in glioma.

Glioblastoma is defined by its aggressive invasion, microvascular proliferation, and central necrosis. BMS-354825 (dasatinib) is an ATP-competitive small-molecule inhibitor effective in treating drug-resistant tumors with mutant BCR-ABL, KIT, and epidermal growth factor receptor by blocking tyrosine phosphorylation sites that are critical in tumorigenesis. In studying the action of dasatinib in human glioblastoma, we found that levels of phospho-SRC, AKT, and ribosomal protein S6 were decreased in cell lines treated with low nanomolar concentrations of dasatinib at baseline and following stimulation with epidermal growth factor. Furthermore, an increased sensitivity to dasatinib was noted in glioma cells with functional PTEN. Reduction of invasive potential was observed in vitro at concentrations well below the IC(50) of dasatinib, which was corroborated by immunofluorescence staining showing disruption of paxillin localization to focal adhesions and decreases in focal adhesion kinase autophosphorylation. Cell cycle analysis revealed minimal G(1) arrest but a significant increase in autophagic cell death in glioma cells treated with dasatinib as assessed by acridine orange staining and a concomitant increase in light chain 3 expression and processing. Combination treatment of glioma cells with dasatinib and temozolomide resulted in a significant increase in cell cycle disruption and autophagic cell death. Dasatinib in combination with temozolomide more effectively increased the therapeutic efficacy of temozolomide than when dasatinib was combined with carboplatin or irinotecan. These results strongly support the clinical use of dasatinib in the treatment of glioblastoma and provide a rationale for combination therapy with dasatinib and temozolomide.

AMPA receptors promote perivascular glioma invasion via beta1 integrin-dependent adhesion to the extracellular matrix.

High-grade gliomas release excitotoxic concentrations of glutamate, which has been shown to enhance tumor proliferation and migration. alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) glutamate receptors are abundantly expressed at the invading edge of glioblastoma specimens, suggesting they may play an important biologic role in tumor invasion. In this study, we examined potential mechanisms by which AMPA receptor (AMPAR) expression and stimulation promote glioma cell migration and invasion. Overexpression of GluR1, the most abundant AMPAR subunit in gliomas, positively correlated with glioma cell adhesion to type I and type IV collagen, which was decreased in cells with knockdown of GluR1 and with blocking antibodies to beta1 integrin. Furthermore, stimulation of the AMPAR led to detachment of cells from the extracellular matrix (ECM). Immunoprecipitation studies showed that GluR1 associated with the actin cytoskeleton-linked protein band 4.1B (brain type), which may serve as a link between GluR1 and integrins. Overexpression of GluR1 correlated with increased cell-surface expression of beta1 integrin, increased phosphorylation of focal adhesion kinase (FAK-Y397), and enhanced numbers of focal adhesion (FA) complexes. Cells overexpressing GluR1 had increased colocalization of actin and paxillin at FAs and, in several glioma cell lines, significantly increased invasion in an in vitro Matrigel transwell assay. Likewise, in an intracranial xenograft model, overexpression of GluR1 led to perivascular and subependymal glioma cell invasion similar to patterns of tumor dissemination described in human glioblastoma. Together, these results suggest that AMPARs may link signals from the ECM to sites of FA, where signal integration promotes tumor invasion.

Endogenous hydrogen peroxide regulates glutathione redox via nuclear factor erythroid 2-related factor 2 downstream of phosphatidylinositol 3-kinase during muscle differentiation.

We reported previously that endogenous reactive oxygen species (ROS) function as myogenic signaling molecules. It has also been determined that excess ROS induce electrophile-response element (EpRE)-driven gene expression via activation of nuclear factor erythroid 2-related factor 2 (Nrf2). Nonetheless, the relationship between the metabolism of ROS (eg, H(2)O(2)) through glutathione (GSH) up-regulation, GSH-dependent reduction of H(2)O(2), and Nrf2-dependent gene regulation is not well established. Therefore, we attempted to determine whether H(2)O(2) controls the intracellular GSH redox state via the Nrf2-glutamate-cysteine ligase (GCL)/glutathione reductase (GR)-GSH signaling pathway. In our experiments, enhanced H(2)O(2) generation was accompanied by an increase in both total GSH levels and the GSH/GSSG ratio during muscle differentiation. Both GCL and GR transcriptional expression levels were markedly increased during muscle differentiation but reduced by catalase treatment. Nrf2 protein expression and nuclear translocation increased during myogenesis. The inhibition of GCL, GR, and Nrf2 both by inhibitors and by RNA interference blocked muscle differentiation. Phosphatidylinositol 3-kinase regulated the expression of the GCL C (a catalytic subunit) and GR genes via the induction of Nrf2 nuclear translocation and expression. In conclusion, endogenous H(2)O(2) generated during muscle differentiation not only functions as a signaling molecule, but also regulates the GSH redox state via activation of the Nrf2-GCL/GR-GSH signaling pathway downstream of phosphatidylinositol 3-kinase.