CXCR4 expression is associated with proneural-to-mesenchymal transition in glioblastoma.

Glioblastoma (GBM) is the most common primary intracranial malignant tumor and consists of three molecular subtypes: proneural (PN), mesenchymal (MES) and classical (CL). Transition between PN to MES subtypes (PMT) is the glioma analog of the epithelial-mesenchymal transition (EMT) in carcinomas and is associated with resistance to therapy. CXCR4 signaling increases the expression of MES genes in glioma cell lines and promotes EMT in other cancers. RNA sequencing (RNAseq) data of PN GBMs in The Cancer Genome Atlas (TCGA) and secondary high-grade gliomas (HGGs) from an internal cohort were examined for correlation between CXCR4 expression and survival as well as expression of MES markers. Publicly available single-cell RNA sequencing (scRNAseq) data was analyzed for cell type specific CXCR4 expression. These results were validated in a genetic mouse model of PN GBM. Higher CXCR4 expression was associated with significantly reduced survival and increased expression of MES markers in TCGA and internal cohorts. CXCR4 was expressed in immune and tumor cells based on scRNAseq analysis. Higher CXCR4 expression within tumor cells on scRNAseq was associated with increased MES phenotype, suggesting a cell-autonomous effect. In a genetically engineered mouse model, tumors induced with CXCR4 exhibited a mesenchymal phenotype and shortened survival. These results suggest that CXCR4 signaling promotes PMT and shortens survival in GBM and highlights its inhibition as a potential therapeutic strategy.

Suppression of RAF/MEK or PI3K synergizes cytotoxicity of receptor tyrosine kinase inhibitors in glioma tumor-initiating cells.

BACKGROUND: The majority of glioblastomas have aberrant receptor tyrosine kinase (RTK)/RAS/phosphoinositide 3 kinase (PI3K) signaling pathways and malignant glioma cells are thought to be addicted to these signaling pathways for their survival and proliferation. However, recent studies suggest that monotherapies or inappropriate combination therapies using the molecular targeted drugs have limited efficacy possibly because of tumor heterogeneities, signaling redundancy and crosstalk in intracellular signaling network, indicating necessity of rationale and methods for efficient personalized combination treatments. Here, we evaluated the growth of colonies obtained from glioma tumor-initiating cells (GICs) derived from glioma sphere culture (GSC) in agarose and examined the effects of combination treatments on GICs using targeted drugs that affect the signaling pathways to which most glioma cells are addicted. METHODS: Human GICs were cultured in agarose and treated with inhibitors of RTKs, non-receptor kinases or transcription factors. The colony number and volume were analyzed using a colony counter, and Chou-Talalay combination indices were evaluated. Autophagy and apoptosis were also analyzed. Phosphorylation of proteins was evaluated by reverse phase protein array and immunoblotting. RESULTS: Increases of colony number and volume in agarose correlated with the Gompertz function. GICs showed diverse drug sensitivity, but inhibitions of RTK and RAF/MEK or PI3K by combinations such as EGFR inhibitor and MEK inhibitor, sorafenib and U0126, erlotinib and BKM120, and EGFR inhibitor and sorafenib showed synergy in different subtypes of GICs. Combination of erlotinib and sorafenib, synergistic in GSC11, induced apoptosis and autophagic cell death associated with suppressed Akt and ERK signaling pathways and decreased nuclear PKM2 and ß-catenin in vitro, and tended to improve survival of nude mice bearing GSC11 brain tumor. Reverse phase protein array analysis of the synergistic treatment indicated involvement of not only MEK and PI3K signaling pathways but also others associated with glucose metabolism, fatty acid metabolism, gene transcription, histone methylation, iron transport, stress response, cell cycle, and apoptosis. CONCLUSION: Inhibiting RTK and RAF/MEK or PI3K could induce synergistic cytotoxicity but personalization is necessary. Examining colonies in agarose initiated by GICs from each patient may be useful for drug sensitivity testing in personalized cancer therapy.

Signal transducer and activator of transcription 5b drives malignant progression in a PDGFB-dependent proneural glioma model by suppressing apoptosis.

Signal transducer and activator of transcription 5b (STAT5b) is likely the relevant STAT5 isoform with respect to the process of malignant progression in gliomas. STAT5b is a latent cytoplasmic protein involved in cell signaling through the modulation of growth factors, apoptosis, and angiogenesis. Previous in vitro studies have shown increased STAT5b expression in glioblastomas relative to low-grade tumors and normal brain. We recently demonstrated that phosphorylated STAT5b associates with delta epidermal growth factor receptor in the nucleus and subsequently binds the promoters of downstream effector molecules, including aurora kinase A. Analysis of TCGA dataset reveals that STAT5b is predominantly expressed in proneural (PN) gliomas relative to mesenchymal and neural gliomas. Here, we modeled ectopic expression of STAT5b in vivo using a platelet-derived growth factor subunit B (PDGFB)-dependent mouse model of PN glioma to determine its effect on tumor formation and progression. We showed that coexpression of STAT5b and PDGFB in mice yielded a significantly higher rate of high-grade gliomas than PDGFB expression alone. We also observed shorter survival in the combined expression set. High-grade tumors from the STAT5b + PDGFB expression set were found to have a lower rate of apoptosis than those from PDGFB alone. Furthermore, we showed that increased expression of STAT5b + PDGFB led to increased expression of downstream STAT5b targets, including Bcl-xL, cyclin D1 and aurora kinase A in high-grade tumors when compared to tumors derived from PDGFB alone. Our findings show that STAT5b promotes the malignant transformation of gliomas, particularly the PN subtype, and is a potential therapeutic target.

Access to the nucleus and functional association with c-Myc is required for the full oncogenic potential of ΔEGFR/EGFRvIII.

ΔEGFR is a potent glioblastoma oncogene which has been studied primarily as a plasma membrane kinase. Using intracranial xenograft studies in mice, we show that blocking ΔEGFR access to the nucleus attenuates its tumorigenicity and, conversely, that promoting nuclear accumulation enhances this, providing the first in vivo evidence that the nuclear actions of ΔEGFR contribute strongly to its oncogenic function. Nuclear actions of ΔEGFR include regulation of gene expression by participation in chromatin-bound complexes, and genome-wide mapping of these sequences by chromatin immunoprecipitation and massively parallel sequencing identified 2294 peaks. Bioinformatic analysis showed enrichment of the E-box motif in the dataset, and c-Myc and ΔEGFR were corecruited to the promoters of and transcriptionally activated a subset of nuclear ΔEGFR chromatin targets. Knockdown of c-Myc decreased the expression of these targets and diminished ΔEGFR-stimulated anchorage-independent colony formation. We conclude that transcriptional regulation of target genes by association with gene regulatory chromatin in cooperation with c-Myc by nuclear ΔEGFR makes a unique contribution to its oncogenicity and propose that this venue provides new targets for therapeutic intervention.

Nuclear EGFRvIII-STAT5b complex contributes to glioblastoma cell survival by direct activation of the Bcl-XL promoter.

Aberrant EGFR signaling strongly promotes glioma malignancy and treatment resistance. The most prevalent mutation, ΔEGFR/EGFRvIII, is an in-frame deletion of the extracellular domain, which occurs in more than 25% of glioblastomas and enhances growth and survival of tumor cells. Paradoxically, the signaling of the potent oncogene ΔEGFR is of low intensity, raising the question of whether it exhibits preferential signaling to key downstream targets. We have observed levels of phosphorylation of STAT5 at position Y699 in cells expressing ΔEGFR that are similar or higher than in cells that overexpress EGFR and are acutely stimulated with EGF, prompting us to investigate the role of STAT5 activation in glioblastoma. Here, we show that in human glioblastoma samples, pSTAT5 levels correlated positively with EGFR expression and were associated with reduced survival. Interestingly, the activation of STAT5b downstream of ΔEGFR was dependent on SFKs, while the signal from acutely EGF-stimulated EGFR to STAT5b involved other kinases. Phosphorylated STAT5b and ΔEGFR associated in the nucleus, bound DNA and were found on promoters known to be regulated by STAT5 including that of the Aurora A gene. ΔEGFR cooperated with STAT5b to regulate the Bcl-XL promoter and knockdown of STAT5b suppressed anchorage independent growth, reduced the levels of Bcl-XL and sensitized glioblastoma cells to cisplatin. Together these results delineate a novel association of nuclear ΔEGFR with STAT5b, which promotes oncogenesis and treatment resistance in glioblastoma by direct regulation of anti-apoptotic gene, Bcl-XL.

Analysis of phosphotyrosine signaling in glioblastoma identifies STAT5 as a novel downstream target of ΔEGFR.

An in-frame deletion mutation in Epidermal Growth Receptor (EGFR), ΔEGFR is a common and potent oncogene in glioblastoma (GBM), promoting growth and survival of cancer cells. This mutated receptor is ligand independent and constitutively active. Its activity is low in intensity and thought to be qualitatively different from acutely ligand stimulated wild-type receptor implying that the preferred downstream targets of ΔEGFR play a significant role in malignancy. To understand the ΔEGFR signal, we compared it to that of a kinase-inactivated mutant of ΔEGFR and wild-type EGFR with shotgun phosphoproteomics using an electron-transfer dissociation (ETD) enabled ion trap mass spectrometer. We identified and quantified 354 phosphopeptides corresponding to 249 proteins. Among the ΔEGFR-associated phosphorylations were the previously described Gab1, c-Met and Mig-6, and also novel phosphorylations including that of STAT5 on Y694/9. We have confirmed the most prominent phosphorylation events in cultured cells and in murine xenograft models of glioblastoma. Pathway analysis of these proteins suggests a preference for an alternative signal transduction pathway by ΔEGFR compared to wild-type EGFR. This understanding will potentially benefit the search for new therapeutic targets for ΔEGFR expressing tumors.

Combined action of the dinuclear platinum compound BBR3610 with the PI3-K inhibitor PX-866 in glioblastoma.

Polynuclear platinum compounds are more effective at killing glioblastoma cells than cisplatin, work by a different mechanism, and typically do not induce high levels of apoptosis at early time points after exposure. Here, we tested the hypothesis that combining BBR3610, the most potent polynuclear platinum, with a phosphoinositide-3-kinase (PI3K) inhibitor would promote apoptosis and enhance the impact on glioblastoma cells. The PI3K pathway is commonly activated in glioblastoma and promotes tumor cell survival, suggesting that its inhibition would make cells more sensitive to cytotoxic agents. We chose PX-866 as a PI3K inhibitor as it is a clinically promising agent being evaluated for brain tumor therapy. Combining BBR3610 and PX-866 resulted in synergistic killing of cultured glioma cells and an extension of survival in an orthotopic xenograft animal model. Both agents alone induced autophagy, and this appeared to be saturated, because when they were combined no additional autophagy was observed. However, the combination of PX-866 and BBR3610 did induce statistically significant increases in the level of apoptosis, associated with a reduction in pAkt and pBad, as well as inhibition of transwell migration. We conclude that combining polynuclear platinums with PI3K inhibitors has translational potential and alters the cellular response to include early apoptosis.

Opening of the Blood-Brain Barrier Using Low-Intensity Pulsed Ultrasound Enhances Responses to Immunotherapy in Preclinical Glioma Models.

PURPOSE: The blood-brain barrier (BBB) inhibits adequate dosing/penetration of therapeutic agents to malignancies in the brain. Low-intensity pulsed ultrasound (LIPU) is a safe therapeutic method of temporary BBB disruption (BBBD) to enhance chemotherapeutic delivery to the tumor and surrounding brain parenchyma for treatment of glioblastoma. EXPERIMENTAL DESIGN: We investigated if LIPU could enhance therapeutic efficacy of anti-PD-1 in C57BL/6 mice bearing intracranial GL261 gliomas, epidermal growth factor receptor variant III (EGFRvIII) chimeric antigen receptor (CAR) T cells in NSG mice with EGFRvIII-U87 gliomas, and a genetically engineered antigen-presenting cell (APC)-based therapy producing the T-cell attracting chemokine CXCL10 in the GL261-bearing mice. RESULTS: Mice treated with anti-PD-1 and LIPU-induced BBBD had a median survival duration of 58 days compared with 39 days for mice treated with anti-PD-1, and long-term survivors all remained alive after contralateral hemisphere rechallenge. CAR T-cell administration with LIPU-induced BBBD resulted in significant increases in CAR T-cell delivery to the CNS after 24 (P < 0.005) and 72 (P < 0.001) hours and increased median survival by greater than 129%, in comparison with CAR T cells alone. Local deposition of CXCL10-secreting APCs in the glioma microenvironment with LIPU enhanced T-cell glioma infiltration during the therapeutic window (P = 0.004) and markedly enhanced survival (P < 0.05). CONCLUSIONS: LIPU increases immune therapeutic delivery to the tumor microenvironment with an associated increase in survival and is an emerging technique for enhancing novel therapies in the brain.

Role of CX3CR1 signaling in malignant transformation of gliomas.

BACKGROUND: Chemokine signaling may contribute to progression of low-grade gliomas (LGGs) by altering tumor behavior or impacting the tumor microenvironment. In this study, we investigated the role of CX3C chemokine receptor 1 (CX3CR1) signaling in malignant transformation of LGGs. METHODS: Ninety patients with LGGs were genotyped for the presence of common CX3CR1 V249I polymorphism and examined for genotype-dependent alterations in survival, gene expression, and tumor microenvironment. A genetically engineered mouse model was leveraged to model endogenous intracranial gliomas with targeted expression of CX3C ligand 1 (CX3CL1) and CX3CR1, individually or in combination. RESULTS: LGG patients who were heterozygous (V/I; n = 43) or homozygous (I/I; n = 2) for the CX3CR1 V249I polymorphism had significantly improved median overall (14.8 vs 9.8 y, P < 0.05) and progression-free survival (8.6 vs 6.5 y, P < 0.05) compared with those with the wild type genotype (V/V; n = 45). Tumors from the V/I + I/I group exhibited significantly decreased levels of CCL2 and MMP9 transcripts, correlating with reduced intratumoral M2 macrophage infiltration and microvessel density. In an immunocompetent mouse model of LGGs, coexpression of CX3CL1 and CX3CR1 promoted a more malignant tumor phenotype characterized by increased microglia/macrophage infiltration and microvessel density, resulting in shorter survival. CONCLUSIONS: CX3CR1 V249I polymorphism is associated with improved overall and progression-free survival in LGGs. CX3CR1 signaling enhances accumulation of tumor associated microglia/macrophages and angiogenesis during malignant transformation.

Anti-PD-1 Induces M1 Polarization in the Glioma Microenvironment and Exerts Therapeutic Efficacy in the Absence of CD8 Cytotoxic T Cells.

PURPOSE: Anti-programmed cell death protein 1 (PD-1) therapy has demonstrated inconsistent therapeutic results in patients with glioblastoma (GBM) including those with profound impairments in CD8 T-cell effector responses. EXPERIMENTAL DESIGN: We ablated the CD8α gene in BL6 mice and intercrossed them with Ntv-a mice to determine how CD8 T cells affect malignant progression in forming endogenous gliomas. Tumor-bearing mice were treated with PD-1 to determine the efficacy of this treatment in the absence of T cells. The tumor microenvironment of treated and control mice was analyzed by IHC and FACS. RESULTS: We observed a survival benefit in immunocompetent mice with endogenously arising intracranial glioblastomas after intravenous administration of anti-PD-1. The therapeutic effect of PD-1 administration persisted in mice even after genetic ablation of the CD8 gene (CD8-/-). CD11b+ and Iba1+ monocytes and macrophages were enriched in the glioma microenvironment of the CD8-/- mice. The macrophages and microglia assumed a proinflammatory M1 response signature in the setting of anti-PD-1 blockade through the elimination of PD-1-expressing macrophages and microglia in the tumor microenvironment. Anti-PD-1 can inhibit the proliferation of and induce apoptosis of microglia through antibody-dependent cellular cytotoxicity, as fluorescently labeled anti-PD-1 was shown to gain direct access to the glioma microenvironment. CONCLUSIONS: Our results show that the therapeutic effect of anti-PD-1 blockade in GBM may be mediated by the innate immune system, rather than by CD8 T cells. Anti-PD-1 immunologically modulates innate immunity in the glioma microenvironment-likely a key mode of activity.

The Role of Fibrinogen-Like Protein 2 on Immunosuppression and Malignant Progression in Glioma.

BACKGROUND: Virtually all low-grade gliomas (LGGs) will progress to high-grade gliomas (HGGs), including glioblastoma, the most common malignant primary brain tumor in adults. A key regulator of immunosuppression, fibrinogen-like protein 2 (FGL2), may play an important role in the malignant transformation of LGG to HGG. We sought to determine the mechanism of FGL2 on tumor progression and to show that inhibiting FGL2 expression had a therapeutic effect. METHODS: We analyzed human gliomas that had progressed from low- to high-grade for FGL2 expression. We modeled FGL2 overexpression in an immunocompetent genetically engineered mouse model to determine its effect on tumor progression. Tumors and their associated microenvironments were analyzed for their immune cell infiltration. Mice were treated with an FGL2 antibody to determine a therapeutic effect. Statistical tests were two-sided. RESULTS: We identified increased expression of FGL2 in surgically resected tumors that progressed from low to high grade (n = 10). The Cancer Genome Atlas data showed that LGG cases with overexpression of FGL2 (n = 195) had statistically significantly shorter survival (median = 62.9 months) compared with cases with low expression (n = 325, median = 94.4 months, P < .001). In a murine glioma model, HGGs induced with FGL2 exhibited a mesenchymal phenotype and increased CD4+ forkhead box P3 (FoxP3)+ Treg cells, implicating immunosuppression as a mechanism for tumor progression. Macrophages in these tumors were skewed toward the immunosuppressive M2 phenotype. Depletion of Treg cells with anti-FGL2 statistically significantly prolonged survival in mice compared with controls (n = 11 per group, median survival = 90 days vs 62 days, P = .004), shifted the phenotype from mesenchymal HGG to proneural LGG, and decreased M2 macrophage skewing. CONCLUSIONS: FGL2 facilitates glioma progression from low to high grade. Suppressing FGL2 expression holds therapeutic promise for halting malignant transformation in glioma.

Maspin mediates increased tumor cell apoptosis upon induction of the mitochondrial permeability transition.

Maspin is a unique serpin with the ability to suppress certain types of malignant tumors. It is one of the few p53-targeted genes involved in tumor invasion and metastasis. With this in mind, we attempted to study the molecular mechanism behind this tumor suppression. Maspin-expressing mammary tumors are more susceptible to apoptosis in both implanted mammary tumors in vivo, a three-dimensional spheroid culture system, as well as in monolayer cell culture under lowered growth factors. Subcellular fractionation shows that a fraction of maspin (in both TM40D-Mp and mutant maspinDeltaN cells) translocates to the mitochondria. This translocation of maspin to the mitochondria is linked to the opening of the permeability transition pore, which in turn causes the loss of transmembrane potential, thus initiating apoptotic degradation. This translocation is absent in the other mutant, maspinDeltaRSL. It fails to cause any loss of membrane potential and also shows decreased caspase 3 levels, proving that translocation to the mitochondria is a key event for this increase in apoptosis by maspin. Suppression of maspin overexpression by RNA interference desensitizes cells to apoptosis. Our data indicate that maspin inhibits tumor progression through the mitochondrial apoptosis pathway. These findings will be useful for maspin-based therapeutic interventions against breast cancer.

Maspin is physically associated with [beta]1 integrin regulating cell adhesion in mammary epithelial cells.

Maspin is a tumor-suppressor serpin (serine protease inhibitor), which inhibits cell invasion and migration. Here, we analyzed maspin function in cell adhesion in nontransformed mammary epithelial cells and investigated the underlying mechanism involved in this process. We report that maspin acts in the early steps in the cell adhesion process. Addition of recombinant maspin rapidly increased MCF-10A cell adhesion to the endogenously deposited matrix, and conversely both an antimaspin antibody (Ab) and maspin knockdown by RNA interference resulted in decreased cell adhesion. Mutation analyses revealed that a region of 86 amino acids located between aa 139 and aa 225 was responsible for maspin effect on adhesion. In addition, we show that maspin is associated with detergent-insoluble cortical cytoskeleton elements. Collectively, these results suggest that maspin is part of the supramolecular structure of the adhesion plaque and it modulates cell adhesion via a beta1 integrin-dependent mechanism.

Analysis of the inhibitors of apoptosis identifies BIRC3 as a facilitator of malignant progression in glioma.

Gliomas, the most common primary brain tumor in humans, include a spectrum of disease. High-grade gliomas (HGG), such as glioblastoma, may arise from low-grade gliomas (LGG) that have a more indolent course. The process of malignant transformation (MT) of LGG to HGG is poorly understood but likely involves the activation of signaling programs that suppress apoptosis. We previously showed that Survivin (BIRC5) plays a role in malignant progression of glioma. Here, we investigated the role of the remaining members of the Inhibitors of Apoptosis (IAP) family on promoting MT in glioma. Utilizing expression data from the cancer genome atlas (TCGA), we identified BIRC3 as a key facilitator of MT from LGG to HGG. TCGA HGGs with high expression of BIRC 3 demonstrated a survival disadvantage and expression levels of BIRC3 were also significantly higher in TCGA HGG compared to TCGA LGG cases. We validated our findings from TCGA by using matched human specimens to show that BIRC expression is increased in HGG compared to their precursor LGG lesions. Using a unique murine model of glioma, we show that overexpression of BIRC3 promotes higher grade glioma and significantly reduces tumor-free survival in mice.

The Duality of Fgl2 - Secreted Immune Checkpoint Regulator Versus Membrane-Associated Procoagulant: Therapeutic Potential and Implications.

Fibrinogen-like protein 2 (Fgl2), a member of the fibrinogen family, can be expressed as a membrane-associated protein with coagulation activity or in a secreted form possessing unique immune suppressive functions. The biological importance of Fgl2 is evident within viral-induced fibrin depositing inflammatory diseases and malignancies and provides a compelling rationale for Fgl2 expression to not only be considered as a disease biomarker but also as a therapeutic target. This article will provide a comprehensive review of the currently known biological properties of Fgl2 and clarifies future scientific directives.

Survivin transcript variant 2 drives angiogenesis and malignant progression in proneural gliomas.

BACKGROUND: The influence of survivin isoforms on outcome in glioblastoma is poorly understood. We analyzed the dominant anti-apoptotic transcript variants of survivin using expression data and modeled them in vivo to determine their impact on glioma formation and progression. METHODS: Using data from low- and high-grade glioma knowledge bases, we expressed the anti-apoptotic isoforms of survivin (transcript variants 1 and 2) in vivo using the RCAS/Ntv-a model of murine glioma. RESULTS: In low-grade gliomas, survivin RNA expression was increased in 22 of 167 (13.2%) of cases and was associated with shortened survival (P = .005). Survivin RNA was preferentially expressed in proneural (PN) relative to mesenchymal high-grade gliomas (P < .0001). In proneural gliomas, survivin was expressed in 94 of 141 (67%) of cases and was associated with shorter disease-free survival (P = .04). In a platelet-derived growth factor subunit B-dependent murine model of PN glioma, ectopic expression of variant 1 yielded tumors in 28 of 30 (93%) of mice, of which 25% were high-grade tumors, whereas ectopic expression of variant 2 yielded tumors in 27 of 28 (96%), of which 81% were high-grade tumors (P < .0001). Microvascular proliferation was significantly more prominent (P < .0001), and tumor-free survival was shorter in mice with variant 2 than variant 1-derived tumors (P = .01). CONCLUSIONS: Survivin expression in low-grade gliomas is associated with poor survival and is preferentially expressed in PN gliomas. Compared with variant 1, variant 2 was associated with poorer survival and promoted malignant progression, angiogenesis, and shorter tumor-free survival in the PN murine model. Inhibiting survivin transcript variant 2, rather than variant 1 (the common isoform), may be an effective treatment strategy for glioma.

Regulation of HGF expression by ΔEGFR-mediated c-Met activation in glioblastoma cells.

The hepatocyte growth factor receptor (c-Met) and a constitutively active mutant of the epidermal growth factor receptor (ΔEGFR/EGFRvIII) are frequently overexpressed in glioblastoma (GBM) and promote tumorigenesis. The mechanisms underlying elevated hepatocyte growth factor (HGF) production in GBM are not understood. We found higher, coordinated mRNA expression levels of HGF and c-Met in mesenchymal (Mes) GBMs, a subtype associated with poor treatment response and shorter overall survival. In an HGF/c-Met-dependent GBM cell line, HGF expression declined upon silencing of c-Met using RNAi or by inhibiting its activity with SU11274. Silencing c-Met decreased anchorage-independent colony formation and increased the survival of mice bearing intracranial GBM xenografts. Consistent with these findings, c-Met activation by ΔEGFR also elevated HGF expression, and the inhibition of ΔEGFR with AG1478 reduced HGF levels. Interestingly, c-Met expression was required for ΔEGFR-mediated HGF production, anchorage-independent growth, and in vivo tumorigenicity, suggesting that these pathways are coupled. Using an unbiased mass spectrometry-based screen, we show that signal transducer and activator of transcription 3 (STAT3) Y705 is a downstream target of c-Met signaling. Suppression of STAT3 phosphorylation with WP1193 reduced HGF expression in ΔEGFR-expressing GBM cells, whereas constitutively active STAT3 partially rescued HGF expression and colony formation in c-Met knockdown cells expressing ΔEGFR. These results suggest that the c-Met/HGF signaling axis is enhanced by ΔEGFR through increased STAT3-dependent HGF expression and that targeting c-Met in Mes GBMs may be an important strategy for therapy.

Forced dimerization increases the activity of ΔEGFR/EGFRvIII and enhances its oncogenicity.

Delta epidermal growth factor receptor (ΔEGFR), an in-frame deletion mutant of the extracellular ligand-binding domain, which occurs in about 30% of glioblastoma, is a potent oncogene that promotes tumor growth and progression. The signaling of ΔEGFR is ligand-independent and low intensity, allowing it to evade the normal mechanisms of internalization and degradation by the endocytic machinery and hence is persistent. The basis of the oncogenic potential of ΔEGFR remains incompletely understood, including whether dimerization plays an important role in its signal and whether its oncogenic potential is dependent on its relatively low intensity, when compared with the acutely activated wild-type receptor. To examine these two important questions, we have generated a chimeric ΔEGFR that allows forced dimerization via domains derived from variants of the FKBP12 protein that are brought together by FK506 derivatives. Forced dimerization of chimeric ΔEGFR significantly increased the intensity of its signal, as measured by receptor phosphorylation levels, suggesting that the naturally occurring ΔEGFR does not form strong or stable dimers as part of its low level signal. Interestingly, the increased activity of dimerized, chimeric ΔEGFR did not promote receptor internalization, implying that reduced rate of endocytic downregulation of ΔEGFR is an inherent characteristic. Significantly, forced dimerization enhanced the oncogenic signal of the receptor, implying that the ΔEGFR is a potent oncogene despite, not because of its low intensity.