epidermal-growth-factor and Glioblastoma

The receptor for epidermal growth factor (EGFR) is a prime target for cancer therapy across a broad variety of tumor types. As it is a tyrosine kinase, small molecule tyrosine kinase inhibitors (TKIs) targeting signal transduction, as well as monoclonal antibodies against the EGFR, have been investigated as anti-tumor agents. However, despite the long-known enigmatic EGFR gene amplification and protein overexpression in glioblastoma, the most aggressive intrinsic human brain tumor, the potential of EGFR as a target for this tumor type has been unfulfilled. This review analyses the attempts to use TKIs and monoclonal antibodies against glioblastoma, with special consideration given to immunological approaches, the use of EGFR as a docking molecule for conjugates with toxins, T-cells, oncolytic viruses, exosomes and nanoparticles. Drug delivery issues associated with therapies for intracerebral diseases, with specific emphasis on convection enhanced delivery, are also discussed.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Brain Neoplasms; Drug Delivery Systems; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Humans; Molecular Targeted Therapy; Protein Kinase Inhibitors

The epidermal growth factor receptor (EGFR) and its ligands figure prominently in the biology of gliomas, the most common tumors of the central nervous system (CNS). Although their histologic classification seems to be straightforward, these tumors constitute a heterogeneous class of related neoplasms. They are associated with a variety of molecular abnormalities affecting signal transduction, transcription factors, apoptosis, angiogensesis, and the extracellular matrix. Under normal conditions, these same interacting factors drive CNS growth and development. We are now recognizing the diverse molecular genetic heterogeneity that underlies tumors classified histologically into three distinct grades. This recognition is leading to new therapeutic strategies targeted directly at specific molecular subtypes. In this article, we will review the role of EGFR and related molecular pathways in the genesis of the normal CNS and their relationship to glial tumorigenesis. We will discuss barriers to effective treatment as they relate to anatomic specialization of the CNS. We will also consider the ways in which specific EGFR alterations common to glioma reflect outcomes following treatment with targeted therapies, all with an eye towards applying this understanding to improved patient outcomes.

Topics: Brain; Brain Neoplasms; Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Gene Expression Regulation, Neoplastic; Glioblastoma; Glioma; Humans; Models, Biological; Multigene Family; Mutation; Protein Binding; Signal Transduction

The role of dysregulated epidermal growth factor receptor-tyrosine kinase (EGFR-TK) activity in promoting tumor resistance to radiation therapy is discussed, and evidence supporting the rationale for the use of gefitinib (IRESSA, ZD1839) to enhance tumor radiosensitivity is reviewed.. A review of the literature regarding the role of EGFR-TK signaling in tumor response to radiation therapy was conducted, and results were summarized from preclinical and clinical studies of gefitinib in the treatment of solid tumors alone and in combination with radiation therapy.. Preclinical results indicate that EGFR-TK activity in tumors can block the cytotoxic effects of radiation therapy and enhance tumor repopulation, resulting in failure of local tumor control. In xenograft tumor models, gefitinib in combination with ionizing radiation resulted in additive to synergistic growth inhibition. In randomized clinical trials, gefitinib has demonstrated efficacy with favorable tolerability as monotherapy for patients with advanced non-small-cell lung cancer or head-and-neck carcinomas who had previously received standard therapies.. These results indicate that there is potential for improved responses by combining gefitinib with radiation therapy in non-small-cell lung cancer, head-and-neck cancers, and other solid tumors.

Topics: Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Combined Modality Therapy; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Glioblastoma; Head and Neck Neoplasms; Humans; Lung Neoplasms; Neoplasm Proteins; Neoplasms; Quinazolines; Radiation Tolerance; Radiation-Sensitizing Agents

Primary brain tumors including anaplastic astrocytomas and glioblastoma multiforme are difficult to treat because of their locally invasive nature and chemoradioresistance. Novel therapies are needed. One class of therapeutics is fusion proteins consisting of peptide toxins fused to brain tumor selective ligands. DAB389EGF is a fusion protein composed of the catalytic and translocation domains of diphtheria toxin fused via a His-Ala linker to human epidermal growth factor (EGF). DAB389EGF is selectively toxic to EGF receptor (EGFR) overexpressing cells. Close to half of all high-grade primary brain tumors have EGFR gene amplification and EGFR overexpression. With the use of convection-enhanced delivery (CED), DAB389EGF may be delivered locally at high concentrations to the brain tumor. CED would avoid many of the pharmacologic and toxicologic barriers which have limited effective use of this agent including rapid clearance from the circulation, high anti-diphtheria toxin antibody titers in the blood and toxicities to the liver and kidney. Both cell lines and animal models are available to assess the potential of this agent for brain tumor therapy. Since significant amounts of clinical grade DAB389EGF are available, some careful additional preclinical efficacy work should lead to testing of this agent in patients within the next few years.

Topics: Animals; Antineoplastic Agents; Diphtheria Toxin; Dose-Response Relationship, Drug; Drug Delivery Systems; Epidermal Growth Factor; Glioblastoma; Humans; Recombinant Fusion Proteins

The understanding of the signal transduction cascade involving growth factors and their receptors is one major key for diagnostic and therapeutic improvements in human neoplasms. Using receptor autoradiography, an inverse relationship for the incidence of somatostatin receptors (SSR) and epidermal growth factor receptors (EGFR) was found in gliomas [1]. In the majority of low grade gliomas, SSR were present but EGFR were absent. In contrast, EGFR were present in most glioblastomas, but no SSR were detected. Recently, the amplification of the EGFR gene and its overexpression was demonstrated to be associated with the development of glioblastomas. Several independent reports revealed that 40-50% of tumors show amplified EGFR [2-4]. The frequency of EGFR amplification was directly associated with tumor malignancy. In addition, amplified EGFR levels indicate a bad prognosis and shorter overall survival [5]. Recent analysis of the EGFR gene in tumors has shown that regions of this gene frequently undergo alteration. Hence, not only amplification but also mutation may be the cause of the increased malignancy in EGFR overexpressing cells [6].

Topics: Brain Neoplasms; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Glioblastoma; Glioma; Growth Substances; Humans; Receptors, Growth Factor; Receptors, Somatostatin; Second Messenger Systems; Signal Transduction

Normal cell replication is regulated by growth factors such as epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) that act through binding to specific surface receptors on target cells. Oncogenes may exert their transforming activity by encoding proteins that mimic the function of the normal regulatory factors along the mitogenic pathway, growth factors, their receptors or elements along the postreceptor signaling system. This may be exemplified by the human malignant glioma, in which the sis gene (encoding a growth factor homologous to PDGF) and the erb B gene (encoding a membrane protein homologous to the EGF receptor) have been implicated.

Topics: Animals; Cell Cycle; Cell Line; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Glioblastoma; Glioma; Growth Substances; Humans; Middle Aged; Mitosis; Oncogenes; Platelet-Derived Growth Factor; Receptors, Cell Surface; Receptors, Platelet-Derived Growth Factor

To evaluate the efficacy and tolerability of gefitinib (ZD1839, Iressa; AstraZeneca, Wilmington, DE), a novel epidermal growth factor receptor tyrosine kinase inhibitor, in patients with recurrent glioblastoma.. This was an open-label, single-center phase II trial. Fifty-seven patients with first recurrence of a glioblastoma who were previously treated with surgical resection, radiation, and usually chemotherapy underwent an open biopsy or resection at evaluation for confirmation of tumor recurrence. Each patient initially received 500 mg of gefitinib orally once daily; dose escalation to 750 mg then 1,000 mg, if a patient received enzyme-inducing antiepileptic drugs or dexamethasone, was allowed within each patient.. Although no objective tumor responses were seen among the 53 assessable patients, only 21% of patients (11 of 53 patients) had measurable disease at treatment initiation. Seventeen percent of patients (nine of 53 patients) underwent at least six 4-week cycles, and the 6-month event-free survival (EFS) was 13% (seven of 53 patients). The median EFS time was 8.1 weeks, and the median overall survival (OS) time from treatment initiation was 39.4 weeks. Adverse events were generally mild (grade 1 or 2) and consisted mainly of skin reactions and diarrhea. Drug-related toxicities were more frequent at higher doses. Withdrawal caused by drug-related adverse events occurred in 6% of patients (three of 53 patients). Although the presence of diarrhea positively predicted favorable OS from treatment initiation, epidermal growth factor receptor expression did not correlate with either EFS or OS.. Gefitinib is well tolerated and has activity in patients with recurrent glioblastoma. Further study of this agent at higher doses is warranted.

Topics: Adult; Aged; Brain Neoplasms; Disease-Free Survival; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epidermal Growth Factor; Female; Gefitinib; Glioblastoma; Humans; Male; Middle Aged; Neoplasm Recurrence, Local; Protein-Tyrosine Kinases; Quinazolines; Treatment Outcome

Topics: Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; Focal Adhesion Protein-Tyrosine Kinases; Glioblastoma; Glycogen Synthase Kinase 3 beta; Humans; Proto-Oncogene Proteins c-akt; Triterpenes

Epithelial membrane protein 3 (EMP3) is an N-glycosylated tetraspanin with a putative trafficking function. It is highly expressed in isocitrate dehydrogenase-wild-type glioblastoma (IDH-wt GBM), and its high expression correlates with poor survival. However, the exact trafficking role of EMP3 and how it promotes oncogenic signaling in GBM remain unclear. Here, we show that EMP3 promotes EGFR/CDK2 signaling by regulating the trafficking and enhancing the stability of EGFR. BioID2-based proximity labeling revealed that EMP3 interacts with endocytic proteins involved in the vesicular transport of EGFR. EMP3 knockout (KO) enhances epidermal growth factor (EGF)-induced shuttling of EGFR into RAB7 + late endosomes, thereby promoting EGFR degradation. Increased EGFR degradation is rescued by the RAB7 negative regulator and novel EMP3 interactor TBC1D5. Phosphoproteomic and transcriptomic analyses further showed that EMP3 KO converges into the inhibition of the cyclin-dependent kinase CDK2 and the repression of EGFR-dependent and cell cycle transcriptional programs. Phenotypically, EMP3 KO cells exhibit reduced proliferation rates, blunted mitogenic response to EGF, and increased sensitivity to the pan-kinase inhibitor staurosporine and the EGFR inhibitor osimertinib. Furthermore, EGFR-dependent patient-derived glioblastoma stem cells display a transcriptomic signature consistent with reduced CDK2 activity, as well as increased susceptibility to CDK2 inhibition upon EMP3 knockdown. Lastly, using TCGA data, we showed that GBM tumors with high EMP3 expression have increased total and phosphorylated EGFR levels. Collectively, our findings demonstrate a novel EMP3-dependent mechanism by which EGFR/CDK2 activity is sustained in GBM. Consequently, EMP3's stabilizing effect provides an additional layer of tumor cell resistance against targeted kinase inhibition.

Topics: Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase 2; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; GTPase-Activating Proteins; Humans; Membrane Glycoproteins; Signal Transduction

The epidermal growth factor receptor (EGFR) is frequently mutated in human cancer

Topics: Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Humans; Ligands; Mutation

Epidermal Growth Factor Receptor (EGFR receptor) is encoded by the EGFR gene. EGFR receptor signaling pathways are activated by EGF protein, regulating cell actions. Overexpression of EGFR receptor may be linked to malignancies with a poor prognosis. As a result, EGFR receptor is being studied for a variety of tumor diagnostics, spurring the development of innovative approaches to increase quality and efficiency. Nanomaterials can recognize cancer cells by specifically targeting of molecular pathways, underscoring the importance of nanomedicine. In this study, we synthesized EGFR-specific nanomarkers by functionalizing EGF protein and Chlorin e6 in gold nanoparticles. These nanoparticles use active targeting to deliver EGF protein to EGFR receptor, and Chlorin e6 serves as a fluorescent marker molecule METHODS: Nanomarkers were examined in vitro in MDA-MB-468 and M059J cell lines. Confocal microscopy and flow cytometry were used to examine the distribution, uptake, internalization, and fluorescence intensity of nanomarkers in vitro RESULTS: The results show that both lines examined accumulate nanomarkers. However, MDA-MB-468 had the highest intensity due to its EGFR receptor overexpression properties CONCLUSION: The findings point to ideal properties for detecting EGFR receptor overexpressed cells.

Topics: Breast Neoplasms; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Female; Glioblastoma; Gold; Humans; Metal Nanoparticles; Photochemotherapy; Triple Negative Breast Neoplasms

The biological role of EGFRvIII (epidermal growth factor receptor variant three) remains unclear.. Three glioblastoma DK-MG sublines were tested with EGF (epidermal growth factor) and TGFβ (transforming growth factor β). Sublines were characterized by an increased percentage of EGFRvIII-positive cells and doubling time (DK-MG. The overexpression of exoEGFRvIII in DK-MG. The roles of TGFβ and EGF in the EGFRvIII context remain unclear. EGFRvIII appears as a weak oncogene and not a marker of GSC (glioma stem cells). Hence, it may not be a proper target for CAR-T (chimeric antigen receptor T cells).

Topics: Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Glioblastoma; Humans; Oncogenes; Proto-Oncogene Proteins c-akt; Receptors, Chimeric Antigen; RNA, Messenger; Transcription Factors; Transforming Growth Factor beta

Conventional approaches for studying and molecular typing of tumors include PCR, blotting, omics, immunocytochemistry, and immunohistochemistry. The last two methods are the most used, as they enable detecting both tumor protein markers and their localizations within the cells. In this study, we have investigated a possibility of using RNA aptamers, in particular, 2'-F-pyrimidyl-RNA aptamer ME07 (48 nucleotides long), specific to the receptor of epidermal growth factor (EGFR, ErbB1, Her1), as an alternative to monoclonal antibodies for aptacytochemistry and aptahistochemistry for human glioblastoma multiforme (GBM). A specificity of binding of FAM-ME07 to the receptor on the tumor cells has been demonstrated by flow cytometry; an apparent dissociation constant for the complex of aptamer - EGFR on the cell has been determined; a number of EGFR molecules has been semi-quantitatively estimated for the tumor cell lines having different amount of EGFR: A431 (10

Topics: Antibodies, Monoclonal; Aptamers, Nucleotide; Brain Neoplasms; Cell Line, Tumor; Cytoplasm; Drug Screening Assays, Antitumor; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Humans; Inhibitory Concentration 50; MCF-7 Cells; Microscopy, Fluorescence; Oligonucleotides; Precision Medicine; Protein Transport; RNA

Overexpression of EGFR drives glioblastomas (GBM) cell invasion but these tumours remain resistant to EGFR-targeted therapies such as tyrosine kinase inhibitors (TKIs). Endocytosis, an important modulator of EGFR function, is often dysregulated in glioma cells and is associated with therapy resistance. However, the impact of TKIs on EGFR endocytosis has never been examined in GBM cells. In the present study, we showed that gefitinib and other tyrosine kinase inhibitors induced EGFR accumulation in early-endosomes as a result of an increased endocytosis. Moreover, TKIs trigger early-endosome re-localization of another membrane receptor, the fibronectin receptor alpha5beta1 integrin, a promising therapeutic target in GBM that regulates physiological EGFR endocytosis and recycling in cancer cells. Super-resolution dSTORM imaging showed a close-proximity between beta1 integrin and EGFR in intracellular membrane compartments of gefitinib-treated cells, suggesting their potential interaction. Interestingly, integrin depletion delayed gefitinib-mediated EGFR endocytosis. Co-endocytosis of EGFR and alpha5beta1 integrin may alter glioma cell response to gefitinib. Using an in vitro model of glioma cell dissemination from spheroid, we showed that alpha5 integrin-depleted cells were more sensitive to TKIs than alpha5-expressing cells. This work provides evidence for the first time that EGFR TKIs can trigger massive EGFR and alpha5beta1 integrin co-endocytosis, which may modulate glioma cell invasiveness under therapeutic treatment.

Topics: Brain Neoplasms; Cell Line, Tumor; Cell Membrane; Cell Movement; Endocytosis; Endosomes; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Glioblastoma; Humans; Integrin alpha5beta1; Protein Kinase Inhibitors; RNA Interference; RNA, Small Interfering

The cystine/glutamate antiporter xCT (SLC7A11) is frequently overexpressed in many cancers, including glioblastoma. Cystine taken up by the cells via xCT is reduced to cysteine, which is used to synthesize glutathione for antioxidant cellular defense. However, overexpression of xCT causes cell death under glucose-limited conditions. We found that stimulation of glioblastoma cells with epidermal growth factor (EGF) induces the upregulation of xCT and promotes cell death under glucose deprivation. Treatment with the mTOR inhibitor Torin 1 suppressed the EGF-induced upregulation of xCT and cell death. EGF increased xCT mRNA levels, which was suppressed by Torin 1. The lysosome inhibitor bafilomycin A1 increased xCT protein levels in the absence of EGF or in the presence of EGF and Torin 1. Taken together, our study suggests that EGF promotes glioblastoma cell death under glucose-limited conditions via the upregulation of xCT at transcriptional and protein levels in an mTOR-dependent manner.

Topics: Amino Acid Transport System y+; Cell Death; Cell Line, Tumor; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Glioblastoma; Glucose; Humans; Neoplasm Proteins; Up-Regulation

Here, a 3D printed multiplexed competitive migration assay is reported for characterizing a chemotactic response in the presence of multiple spatially distributed chemoattractants. The utility of the assay is demonstrated by examining the chemotactic response of human glioblastoma cells to spatially opposing chemotactic gradients of epidermal growth factor (EGF) and bradykinin (BK). Competitive migration assays involving spatially opposing gradients of EGF and BK that are optimized in the absence of the second chemoattractant show that 46% more glioblastoma cells migrate toward EGF sources. The migration velocities of human glioblastoma cells toward EGF and BK sources are reduced by 7.6 ± 2.2% and 11.6 ± 6.3% relative to those found in the absence of the spatially opposing chemoattractant. This work provides new insight to the chemotactic response associated with glioblastoma-vasculature interactions and a versatile, user-friendly platform for characterizing the chemotactic response of cells in the presence of multiple spatially distributed chemoattractants.

Topics: Bradykinin; Cell Line, Tumor; Cell Migration Assays; Chemotactic Factors; Chemotaxis; Epidermal Growth Factor; Equipment Design; Glioblastoma; Humans; Microfluidic Analytical Techniques; Printing, Three-Dimensional

Abnormal secretion of epidermal growth factor (EGF) by non-neuronal cells (e.g., glioma-associated microglia) establishes a feedback loop between glioblastoma multiforme (GBM) invasion and a functional disruption of brain tissue. Considering the postulated significance of this vicious circle for GBM progression, we scrutinized mechanisms of EGF-dependent pro-invasive signaling in terms of its interrelations with energy metabolism and reactive oxygen species (ROS) production. The effects of EGF on the invasiveness of human glioblastoma T98G cells were estimated using time-lapse video microscopy, immunocytochemistry, cell cycle assay, immunoblot analyses, and Transwell® assay. These techniques were followed by quantification of the effect of EGFR (Epidermal Growth Factor Receptor) and ROS inhibitors on the EGF-induced T98G invasiveness and intracellular ROS, ATP, and lactate levels and mitochondrial metabolism. The EGF remarkably augmented the proliferation and motility of the T98G cells. Responses of these cells were accompanied by cellular rear-front polarization, translocation of vinculin to the leading lamellae, and increased promptness of penetration of micropore barriers. Erlotinib (the EGFR inhibitor) significantly attenuated the EGF-induced T98G invasiveness and metabolic reprogramming of the T98G cells, otherwise illustrated by the increased mitochondrial activity, glycolysis, and ROS production in the EGF-treated cells. In turn, ROS inhibition by N-acetyl-L-cysteine (NAC) had no effect on T98G morphology, but considerably attenuated EGF-induced cell motility. Our data confirmed the EGFR/ROS-dependent pro-neoplastic and pro-invasive activity of EGF in human GBM. These EGF effects may depend on metabolic reprogramming of GBM cells and are executed by alternative ROS-dependent/-independent pathways. The EGF may thus preserve bioenergetic homeostasis of GBM cells in hypoxic regions of brain tissue.

Topics: Antineoplastic Agents; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Glioblastoma; Humans; Mitochondria; Reactive Oxygen Species; Signal Transduction

Glioblastoma multiforme (GBM) is the most common malignant tumour in the adult brain and hard to treat. Nuclear factor κB (NF-κB) signalling has a crucial role in the tumorigenesis of GBM. EGFR signalling is an important driver of NF-κB activation in GBM; however, the correlation between EGFR and the NF-κB pathway remains unclear. In this study, we investigated the role of mucosa-associated lymphoma antigen 1 (MALT1) in glioma progression and evaluated the anti-tumour activity and effectiveness of MI-2, a MALT1 inhibitor in a pre-clinical GBM model. We identified a paracaspase MALT1 that is involved in EGFR-induced NF-kB activation in GBM. MALT1 deficiency or inhibition significantly affected the proliferation, survival, migration and invasion of GBM cells both in vitro and in vivo. Moreover, MALT1 inhibition caused G1 cell cycle arrest by regulating multiple cell cycle-associated proteins. Mechanistically, MALTI inhibition blocks the degradation of IκBα and prevents the nuclear accumulation of the NF-κB p65 subunit in GBM cells. This study found that MALT1, a key signal transduction cascade, can mediate EGFR-induced NF-kB activation in GBM and may be potentially used as a novel therapeutic target for GBM.

Topics: Animals; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Humans; Mice, Knockout; Molecular Targeted Therapy; Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein; Neoplasm Invasiveness; NF-kappa B; Tumor Stem Cell Assay

Hypoxia-inducible factor 1α (HIF1α) promotes the malignant progression of glioblastoma under hypoxic conditions, leading to a poor prognosis for patients with glioblastoma; however, none of the therapies targeting HIF1α in glioblastoma have successfully eradicated the tumour. Therefore, we focused on the reason and found that treatments targeting HIF1α and HIF2α simultaneously increased tumour volume, but the combination of HIF1α/HIF2α-targeted therapies with temozolomide (TMZ) reduced tumourigenesis and significantly improved chemosensitization. Moreover, miR-210-3p induced HIF1α expression but inhibited HIF2α expression, suggesting that miR-210-3p regulates HIF1α/HIF2α expression. Epidermal growth factor (EGF) has been shown to upregulate HIF1α expression under hypoxic conditions. However, in the present study, in addition to the signalling pathways mentioned above, the upstream proteins HIF1α and HIF2α have been shown to induce EGF expression by binding to the sequences AGGCGTGG and GGGCGTGG. Briefly, in a hypoxic microenvironment the HIF1α/HIF2α-miR210-3p network promotes the malignant progression of glioblastoma through a positive feedback loop with EGF. Additionally, differentiated glioblastoma cells underwent dedifferentiation to produce glioma stem cells under hypoxic conditions, and simultaneous knockout of HIF1α and HIF2α inhibited cell cycle arrest but promoted proliferation with decreased stemness, promoting glioblastoma cell chemosensitization. In summary, both HIF1α and HIF2α regulate glioblastoma cell proliferation, dedifferentiation and chemoresistance through a specific pathway, which is important for glioblastoma treatments.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Brain Neoplasms; Cell Differentiation; Cell Hypoxia; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; Gene Knockout Techniques; Glioblastoma; Heterografts; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Mice; Mice, Inbred BALB C; MicroRNAs; Signal Transduction

Neural progenitor cells (NPCs) are self-renewing cells that give rise to the major cells in the nervous system and are considered to be the possible cell of origin of glioblastoma. The gap junction protein connexin43 (Cx43) is expressed by NPCs, exerting channel-dependent and -independent roles. We focused on one property of Cx43-its ability to inhibit Src, a key protein in brain development and oncogenesis. Because Src inhibition is carried out by the sequence 266-283 of the intracellular C terminus in Cx43, we used a cell-penetrating peptide containing this sequence, TAT-Cx43

Topics: Animals; Astrocytes; beta Catenin; Carcinogenesis; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell-Penetrating Peptides; Connexin 43; Disease Models, Animal; Epidermal Growth Factor; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Neoplastic Stem Cells; Neural Stem Cells; Rats; src-Family Kinases; Stem Cells

Glioblastoma multiforme (GBM) is one of the most common and deadly cancers of the central nervous system (CNS). It is characterized by the presence of hypoxic regions, especially in the core, leading to an increase in vascularity. This increased vascularization is driven by the expression of the major angiogenic inducer VEGF and the indirect angiogenic inducer Epidermal growth factor (EGF), which stimulates VEGF expression. In this study, we examine the regulation of VEGF by both hypoxia and the EGF signaling pathway. We also examine the involvement of pathways downstream from EGF signaling, including the mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK) pathway and the Phosphatidylinositol-3-kinase/RhoA/C (PI3K/RhoA/C) pathway in this regulation. Our results show that VEGF expression and secretion levels increase following either hypoxia or EGF stimulation, with the two stimuli signaling in parallel. We also observed an increase in ERK and protein kinase B (Akt) phosphorylation, in response to EGF stimulation, with kinetics that correlated with the kinetics of the effect on VEGF. Using pharmacological inhibitors against ERK and PI3K and small interfering RNAs (siRNAs) against RhoA and RhoC, we found that both the ERK and the PI3K/RhoA/C pathways have to cooperate in order to lead to an increase in VEGF expression, downstream from EGF. In response to hypoxia, however, only ERK was involved in the regulation of VEGF. Hypoxia also led to a surprising decrease in the activation of PI3K and RhoA/C. Finally, the decrease in the activation of these Rho-GTPases was found to be mediated through a hypoxia-driven overexpression of the Rho-GTPase GTPase activating protein (GAP), StarD13. Therefore, while under normoxic conditions, EGF stimulates the activation of both the PI3K and the MAPK pathways and the induction of VEGF, in glioblastoma cells, hypoxic conditions lead to the suppression of the PI3K/RhoA/C pathway and an exclusive switch to the MAPK pathway.

Topics: Cell Line, Tumor; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Glioblastoma; GTP Phosphohydrolases; GTPase-Activating Proteins; Humans; Hypoxia; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphorylation; Rho Factor; Signal Transduction; Vascular Endothelial Growth Factor A

EphA3, a member of the Eph family of receptor tyrosine kinases, has been reported to be overexpressed in some human cancers including glioblastoma. Here, we found that expression of EphA3 is up-regulated in response to epidermal growth factor (EGF) stimulation and promotes formation of cell aggregates in suspension culture of glioblastoma cells. Suppression of EphA3 expression by short hairpin RNA-mediated knockdown or CRISPR/Cas9-mediated gene deletion inhibited EGF-induced promotion of cell aggregate formation, whereas overexpression of EphA3 promoted formation of cell aggregates in suspension culture. EGF-induced EphA3 expression and promotion of cell aggregate formation required Akt activity. Furthermore, N-cadherin, whose expression was regulated by EGF and EphA3, contributed to the formation of cell aggregates in suspension culture. These results suggest that the regulation of EphA3 expression plays a critical role in glioblastoma cell growth in non-adherent conditions.

Topics: Brain Neoplasms; Cadherins; Cell Aggregation; Cell Line, Tumor; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Proto-Oncogene Proteins c-akt; Receptor Protein-Tyrosine Kinases; Receptor, EphA3; Suspensions; Up-Regulation

Glioblastoma is the most frequent primary brain tumor in adults and a highly lethal malignancy with a median survival of about 15 months. The aggressive invasion of the surrounding normal brain makes complete surgical resection impossible, increases the resistance to radiation and chemotherapy, and assures tumor recurrence. Thus, there is an urgent need to develop innovative therapeutics to target the invasive tumor cells for improved treatment outcomes of this disease. Expression of TROY (TNFRSF19), a member of the tumor necrosis factor (TNF) receptor family, increases with increasing glial tumor grade and inversely correlates with patient survival. Increased expression of TROY stimulates glioblastoma cell invasion

Topics: Binding Sites; Brain Neoplasms; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Receptors, Tumor Necrosis Factor; Signal Transduction; Up-Regulation

The HMG-box protein 1 (HBP1) is a transcriptional regulator and a potential tumor suppressor that controls cell proliferation, differentiation and oncogene-mediated senescence. In a previous study, we showed that AKT activation through the PI3K/AKT/FOXO pathway represses HBP1 expression at the transcriptional level in human fibroblasts as well as in cancer cell lines. In the present study, we investigated whether AKT could also regulate HBP1 directly. First, AKT1 phosphorylated recombinant human HBP1 in vitro on three conserved sites, Ser380, Thr484 and Ser509. In living cells, we confirmed the phosphorylation of HBP1 on residues 380 and 509 using phospho-specific antibodies. HBP1 phosphorylation was induced by growth factors, such as EGF or IGF-1, which activated AKT. Conversely, it was blocked by treatment of cells with an AKT inhibitor (MK-2206) or by AKT knockdown. Next, we observed that HBP1 transcriptional activity was strongly modified by mutating its phosphorylation sites. The regulation of target genes such as DNMT1, P47phox, p16

Topics: Cell Proliferation; Cellular Senescence; Epidermal Growth Factor; Glioblastoma; HEK293 Cells; High Mobility Group Proteins; Humans; Insulin-Like Growth Factor I; Phosphorylation; Proto-Oncogene Proteins c-akt; Repressor Proteins; Transcription, Genetic

Numerous studies have shown that calmodulin (CaM) is a major regulator of calcium-dependent signaling, which regulates cell proliferation, programmed cell death, and autophagy in cancer. However, limited information is available on mechanisms underlying the effect of CaM on the invasive property of glioblastoma multiforme (GBM) cells, especially with respect to invadopodia formation. In this study, we find that CaM serves as a prognostic factor for GBM, and it is strongly associated with the invasive nature of this tumor. Results of preliminary experiments indicated that CaM concentration was significantly correlated with the invasive capacity of and invadopodia formation by different GBM cell lines. CaM inhibition via a small hairpin RNA or a pharmacological inhibitor significantly disrupted invadopodia formation and MMP activity and downregulated vimentin expression. Moreover, CaM knockdown exerted a strong anti-invasive effect on GBM in vivo. Interestingly, epidermal growth factor treatment promoted CaM redistribution from the nucleus to the cytoplasm, eventually activating invadopodia-associated proteins by binding to them via their cytosolic-binding sites. Moreover, CaM inhibition suppressed the activation of invadopodia-associated proteins. Thus, our findings provide a novel therapeutic strategy to impede GBM invasion by inhibiting invadopodia formation, and shed light on the spatial organization of CaM signals during GBM invasion.

Topics: Brain Neoplasms; Calcium; Calmodulin; Cell Line, Tumor; Epidermal Growth Factor; Glioblastoma; Humans; Neoplasm Invasiveness; Podosomes; Protein Transport; RNA, Small Interfering; Vimentin

Epidermal growth factor family of receptor tyrosine kinases (ERBB) family cell surface receptors, including epidermal growth factor receptor (EGFR/ERBB1), are phosphorylated upon binding by various EGF family ligands and signal via multiple kinase pathways. EGFR signaling is enhanced because of mutational activation of EGFR in almost half of glioblastomas, the most common malignant primary brain tumor. Therapeutic targeting of EGFR in glioblastoma has remained largely unsuccessful. Here, we profiled nine long-term (LTC) and five glioma-initiating (GIC) cell lines for expression and activation of ERBB family receptors and expression of their ligands. Receptors and ligands were abundantly expressed, with patterns overall similar to glioblastoma expression profiles in vivo as deposited in The Cancer Genome Atlas database. No differences between LTC and GIC emerged. Irrespective of ligand or receptor expression, neither an EGFR antibody, erbitux, nor an EGFR tyrosine kinase inhibitor, gefitinib, were particularly active against LTC or GIC at clinically relevant concentrations. Self-renewal capacity of GIC was severely compromised by epidermal growth factor (EGF) withdrawal, but rescued by transforming growth factor alpha (TGF-α), although not by neuregulin-1 (NRG-1). Subcellular fractionation indicated high levels of nuclear phosphorylated EGFR in all LTC and GIC. In LN-229 cells, pERBB2 and pERBB3 were also detected in the nucleus. Nuclear pERBB2 was less sensitive, whereas pERBB3 was induced, in response to gefitinib. This study provides an extensive characterization of human glioma cell models, including stem-like models, with regard to ERBB receptor/ligand expression and signaling. Redundant signaling involving multiple ERBB family ligands and receptors may contribute to the challenges of developing more effective EGFR-targeted therapies for glioblastoma.

Topics: Antineoplastic Agents; Brain Neoplasms; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cetuximab; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Glioblastoma; Humans; Ligands; Transforming Growth Factor alpha

Glioblastoma multiforme (GBM) is the most malignant central nervous system tumor. Alkylating agent, temozolomide (TMZ), is currently the first-line chemotherapeutic agent for GBM. However, the sensitivity of GBM cells to TMZ is affected by many factors. And, several clinic trials, including co-administration of TMZ with other drugs, have failed in successful treatment of GBM. We have previously reported that Netrin-4 (NTN4), a laminin-like axon guidance protein, plays a protective role in GBM cell senescence upon TMZ-triggered DNA damage. However, the master regulator of NTN4 needs further elucidation. Epidermal growth factor/Epidermal growth factor receptor (EGF/EGFR) can modulate the expression of various extracellular matrix related molecules, and prevent DNA damage in GBM cells. In this study, we investigated the relationship between EGF/EGFR signaling and NTN4, and explored their effect on therapeutic efficacy in GBM cells upon TMZ treatment.. Co-expression analysis were performed by using the RNA sequencing data from NIH 934 cell lines and from single cell RNA sequencing data of GBM tumor. The co-expressing genes were used for GO enrichment and signaling pathway enrichment. mRNA expression of the target genes were quantified by qPCR, and cell senescence were investigated by Senescence-Associated Beta-Galactosidase Staining. Protein phosphorylation were observed and analyzed by immunoblotting. The RNA sequencing data and clinical information of TMZ treated patients were extracted from TCGA-glioblastoma project, and then used for Kaplan-Meier survival analysis.. Analysis of RNA sequencing data revealed a potential co-expression relationship between NTN4 and EGFR. GO enrichment of EGFR-correlated genes indicated that EGFR regulates GBM cells in a manner similar to that in central nervous system development and neural cell differentiation. Pathway analysis suggested that EGFR and its related genes contribute to cell adhesion, extracellular matrix (ECM) organization and caspase related signaling. We also show that EGF stimulates NTN4 expression in GBM cells and cooperates with NTN4 to attenuate GBM cell senescence induced by DNA damage, possibly via AKT and ERK. Clinical analysis showed that co-expression of EGFR and NTN4 significantly predicts poor survival in TMZ-treated GBM patients.. This study indicates that EGF/EGFR regulates and cooperates with NTN4 in DNA damage resistance in GBM. Therefore, our findings provide a potential therapeutic target for GBM.

Topics: Brain Neoplasms; Cell Line, Tumor; Cellular Senescence; DNA Damage; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Humans; Netrins; Up-Regulation

The extent of 5-aminolevulinic acid (5-ALA) guided tumor resection has a determining impact in high-grade glioma and glioblastoma surgery. Yet the intensity of the 5-ALA induced fluorescence may vary within the tumor. We aimed to correlate 5-ALA induced fluorescence with the expression of epithelial growth factor receptor (EGFR) and its constitutively active version EGFRvIII in different glioblastoma (GBM) cell lines. To elucidate the role of EGFR in the metabolism of 5-ALA in GBM cell lines with variable EGFR expression status, we analyzed the activation of EGFR by its primary ligand EGF, and its downstream effect on Heme oxygenase-1 (HO-1), a key enzyme regulating the metabolism of Protoporphyrin IX (PpIX), the fluorescent metabolite of 5-ALA. Effects of direct pharmacological inhibition by Tin(IV)-Protoporphyrin (SnPP) or gene knockdown by small interfering RNA (siRNA) on HO-1 enzyme were analyzed in respect to 5-ALA induced fluorescence. Furthermore, inhibition of EGFR by Gefitinib was tested. A significant difference in 5-ALA induced fluorescence was obtained in U87MG (low EGFR expression) and LN229EGFR cells (EGFR overexpression) compared to BS153 (EGFR overexpression/EGFRvIII+). Treatment of U87MG and LN229EGFR cells with EGF significantly reduced cellular fluorescence, by promoting HO-1 transcription and expression in a concentration-dependent manner. This effect could be reversed by EGFR-specific siRNA treatment, which reduced protein expression of about 80% in U87MG. Remarkably, inhibition of HO-1 activity by SnPP or reduction of HO-1 protein levels by siHO-1 treatment restored fluorescence in all cell lines, independently of EGFR quantitative and qualitative expression. Gefitinib treatment was able to restore fluorescence after EGF stimulation in U87MG cells but not in BS153 cells, overexpressing EGFR/EGFRvIII. In GBM cell lines, 5-ALA induced fluorescence is variable and influenced by EGF-induced downstream activation of HO-1. HO-1 protein expression was identified as a negative regulator of 5-ALA induced fluorescence in GBM cells. We further propose that co-expression of EGFRvIII but not quantitative EGFR expression influence HO-1 activity and therefore cellular fluorescence.

Topics: Aminolevulinic Acid; Astrocytes; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Fluorescent Dyes; Gefitinib; Gene Expression; Glioblastoma; Heme Oxygenase-1; Humans; Protein Kinase Inhibitors; Quinazolines; RNA, Messenger

Magnetic nanoparticles (MNPs) functionalized with targeting moieties can recognize specific cell components and induce mechanical actuation under magnetic field. Their size is adequate for reaching tumors and targeting cancer cells. However, due to the nanometric size, the force generated by MNPs is smaller than the force required for largely disrupting key components of cells. Here, we show the magnetic assembly process of the nanoparticles inside the cells, to form elongated aggregates with the size required to produce elevated mechanical forces. We synthesized iron oxide nanoparticles doped with zinc, to obtain high magnetization, and functionalized with the epidermal growth factor (EGF) peptide for targeting cancer cells. Under a low frequency rotating magnetic field at 15 Hz and 40 mT, the internalized EGF-MNPs formed elongated aggregates and generated hundreds of pN to dramatically damage the plasma and lysosomal membranes. The physical disruption, including leakage of lysosomal hydrolases into the cytosol, led to programmed cell death and necrosis. Our work provides a novel strategy of designing magnetic nanomedicines for mechanical destruction of cancer cells.

Topics: Cell Death; Cell Line, Tumor; Epidermal Growth Factor; Ferric Compounds; Glioblastoma; Humans; Magnetic Field Therapy; Metal Nanoparticles; Molecular Targeted Therapy

Glioblastoma multiforme (GBM) is the most common and lethal brain tumor in adults. It is known that amplification of the epidermal growth factor receptor gene (EGFR) occurs in approximately 40% of GBM, leading to enhanced activation of the EGFR signaling pathway and promoting tumor growth. Although GBM mutations are stably maintained in GBM in vitro models, rapid loss of EGFR gene amplification is a common observation during cell culture. To maintain EGFR amplification in vitro, heterotopic GBM xenografts with elevated EGFR copy number were cultured under varying serum conditions and EGF concentrations. EGFR copy numbers were assessed over several passages by quantitative PCR and chromogenic in situ hybridization. As expected, in control assays with 10% FCS, cells lost EGFR amplification with increasing passage numbers. However, cells cultured under serum free conditions stably maintained elevated copy numbers. Furthermore, EGFR protein expression positively correlated with genomic amplification levels. Although elevated EGFR copy numbers could be maintained over several passages in vitro, levels of EGFR amplification were variable and dependent on the EGF concentration in the medium. In vitro cultures of GBM cells with elevated EGFR copy number and corresponding EGFR protein expression should prove valuable preclinical tools to gain a better understanding of EGFR driven glioblastoma and assist in the development of new improved therapies.

Topics: Animals; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Mice

The failure of therapies targeting tumor angiogenesis may be caused by anti-angiogenic resistance mechanisms induced by VEGF and non-VEGF pathways alterations. Anti-angiogenic therapy failure is also attributed to immune system, acting by tumor-associated macrophages that release pro-angiogenic factors and a consequent increase of blood vessels. Recently, in a study by Rheal et al., a new angiogenic receptor, epidermal growth factor, latrophilin, and 7 trans-membrane domain-containing protein 1 on chromosome 1(ELTD1) has been identified as a promising glioma biomarker. In this study we aim to analyse whether this receptor may be used as a target molecule in glioblastoma therapy. Our results showed that small interfering RNA silencing ELTD1 caused cytotoxicity in glioblastoma cells. We also found that PDGFR, VEGFR, and their common PI3K/mTOR intracellular pathway inactivation-induced cytotoxicity in glioblastoma cells. Further, we found high percent of cytotoxicity in a low passage glioblastoma cell line after BEZ235 (a dual inhibitor of PI3K/mTOR pathway) treatment at nanomolar concentrations, compared to AG1433 (a PDGFR inhibitor) and SU1498 (a VEGFR inhibitor) that were only cytotoxic at micromolar ranges. In the future, these could prove as attractive therapeutic targets in single therapy or coupled with classic therapeutic approaches such as chemotherapy of radiotherapy.

Topics: Biomarkers, Tumor; Cell Death; Epidermal Growth Factor; Gene Silencing; Glioblastoma; Humans; Receptors, G-Protein-Coupled; Receptors, Peptide; RNA, Small Interfering

EphA2, a member of the Eph receptor tyrosine kinases, is frequently overexpressed in a variety of malignancies, including glioblastoma, and its expression is correlated with poor prognosis. EphA2 acts as a tumor promoter through a ligand ephrin-independent mechanism, which requires phosphorylation of EphA2 on serine 897 (S897), leading to increased cell migration and invasion. In this study, we show that ligand-independent EphA2 signaling occurs downstream of the MEK/ERK/RSK pathway and mediates epidermal growth factor (EGF)-induced cell proliferation in glioblastoma cells. Suppression of EphA2 expression by long-term exposure to ligand ephrinA1 or EphA2-targeted shRNA inhibited EGF-induced cell proliferation. Stimulation of the cells with EGF induced EphA2 S897 phosphorylation, which was suppressed by MEK and RSK inhibitors, but not by phosphatidylinositol 3-kinase (PI3K) and Akt inhibitors. The RSK inhibitor or RSK2-targeted shRNA also suppressed EGF-induced cell proliferation. Furthermore, overexpression of wild-type EphA2 promoted cell proliferation without EGF stimulation, whereas overexpression of EphA2-S897A mutant suppressed EGF- or RSK2-induced proliferation. Taken together, these results suggest that EphA2 is a key downstream target of the MEK/ERK/RSK signaling pathway in the regulation of glioblastoma cell proliferation.

Topics: Brain Neoplasms; Bromodeoxyuridine; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; Glioblastoma; Humans; MAP Kinase Signaling System; Phosphorylation; Phosphoserine; Receptor, EphA2; Ribosomal Protein S6 Kinases, 90-kDa; Signal Transduction

Extensive heterogeneity is a defining hallmark of glioblastoma multiforme (GBM) at the cellular and molecular levels. EGFRvIII, the most common EGFR mutant, is expressed in 24-67% of cases and strongly indicates a poor survival prognosis. By co-expressing EGFRvIII and EGFRwt, we established an EGFRvIII/wt heterogenic model. Using this approach, we confirmed that a mixture of EGFRvIII and EGFRwt at a certain ratio could clearly enhance tumor growth in vitro and in vivo compared with EGFRwt cells, thereby indicating that EGFRvIII cells promote tumor growth. Furthermore, we demonstrated that the EGFRvIII cells could support the growth of EGFRwt cells by secreting growth factors, thus acting as the principal source for maintaining tumor survival. F25P preproinsulin effectively reduced the concentrations of EGF, VEGF, and MMP-9 in the blood of tumor-bearing mice by competitively inhibiting the endoplasmic reticulum signal peptidase and increased the overall survival in orthotopic models. Taken together, our results provided an effective therapy of F25P preproinsulin in the EGFRvIII/wt heterogenic model.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Genetic Therapy; Glioblastoma; Humans; Insulin; Matrix Metalloproteinase 9; Mice, Inbred BALB C; Mice, Nude; Mutation; Protein Precursors; Signal Transduction; Time Factors; Transfection; Tumor Burden; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Many types of human tumour cells overexpress the dual-specificity phosphatase Cdc25A. Cdc25A dephosphorylates cyclin-dependent kinase and regulates the cell cycle, but other substrates of Cdc25A and their relevant cellular functions have yet to be identified. We demonstrate here that EGFR activation results in c-Src-mediated Cdc25A phosphorylation at Y59, which interacts with nuclear pyruvate kinase M2 (PKM2). Cdc25A dephosphorylates PKM2 at S37, and promotes PKM2-dependent β-catenin transactivation and c-Myc-upregulated expression of the glycolytic genes GLUT1, PKM2 and LDHA, and of CDC25A; thus, Cdc25A upregulates itself in a positive feedback loop. Cdc25A-mediated PKM2 dephosphorylation promotes the Warburg effect, cell proliferation and brain tumorigenesis. In addition, we identify positive correlations among Cdc25A Y59 phosphorylation, Cdc25A and PKM2 in human glioblastoma specimens. Furthermore, levels of Cdc25A Y59 phosphorylation correlate with grades of glioma malignancy and prognosis. These findings reveal an instrumental function of Cdc25A in controlling cell metabolism, which is essential for EGFR-promoted tumorigenesis.

Topics: Animals; beta Catenin; Brain Neoplasms; Carcinogenesis; cdc25 Phosphatases; Cell Line, Tumor; Cell Nucleus; Epidermal Growth Factor; Glioblastoma; Glycolysis; Humans; Mice; Phosphorylation; Phosphoserine; Phosphotyrosine; Prognosis; Protein Binding; Proto-Oncogene Proteins c-myc; Pyruvate Kinase; src-Family Kinases; Transcriptional Activation

Epidermal growth factor receptor (EGFR)vIII is the most common EGFR mutant found in glioblastoma (GBM). EGFRvIII does not bind ligand, is highly oncogenic and is usually coexpressed with EGFR wild type (EGFRwt). EGFRvIII activates Met, and Met contributes to EGFRvIII-mediated oncogenicity and resistance to treatment. Here, we report that addition of EGF results in a rapid loss of EGFRvIII-driven Met phosphorylation in glioma cells. Met is associated with EGFRvIII in a physical complex. Addition of EGF results in a dissociation of the EGFRvIII-Met complex with a concomitant loss of Met phosphorylation. Consistent with the abrogation of Met activation, addition of EGF results in the inhibition of EGFRvIII-mediated resistance to chemotherapy. Thus, our study suggests that ligand in the milieu of EGFRvIII-expressing GBM cells is likely to influence the EGFRvIII-Met interaction and resistance to treatment, and highlights a novel antagonistic interaction between EGFRwt and EGFRvIII in glioma cells.

Topics: Brain Neoplasms; Cell Line, Tumor; Dacarbazine; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Phenotype; Phosphorylation; Proto-Oncogene Proteins c-met; Receptor Protein-Tyrosine Kinases; Temozolomide

CD109 is a glycosylphosphatidylinositol-anchored cell surface protein that is frequently detected in squamous cell carcinomas. CD109 is a negative regulator of TGF-β1 signaling in human keratinocytes, and the N-terminal fragment of CD109 secreted from cells after cleavage by the furin protease is important for modulating TGF-β1 signaling. Previously, we found that CD109 is expressed in human glioblastoma cells; however, the role of CD109 in glioblastoma cells is not established. Here, we describe the effects of CD109 in human glioblastoma cell lines. Three glioblastoma cell lines, SK-MG-1, U251MG and MG178, were tested and CD109 overexpression attenuated TGF-β1 signaling and enhanced EGF signaling in SK-MG-1, but not in U251MG or MG178. The N-terminal CD109 fragment in SK-MG-1 was hyperglycosylated compared with that in MG178 or U251MG. The conditioned medium of CD109-overexpressing SK-MG-1, containing the secreted N-terminal CD109, had a negative effect on TGF-β1 signaling in wild-type SK-MG-1 and MG178, whereas it did not show any effect on EGF signaling. In addition, cell surface CD109 interacts with EGF receptor in SK-MG-1 overexpressing CD109, and exhibited enhanced cell migration and invasion. These findings suggest that CD109 attenuates TGF-β1 signaling and enhances EGF signaling in SK-MG-1 cells and that the membrane-anchored CD109 may play major roles in the EGF signaling pathway.

Topics: Antigens, CD; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Glycosylation; GPI-Linked Proteins; Humans; Keratinocytes; Neoplasm Invasiveness; Neoplasm Proteins; Peptide Fragments; Recombinant Proteins; Signal Transduction; Transforming Growth Factor beta1

As the most aggressive malignant primary human brain tumor, glioblastoma is noted with extremely poor patient survival. Previous studies have demonstrated that expression of matrix metalloproteinase-9 (MMP9) in glioblastoma cells is critical for cancer metastasis. However, the molecular signaling pathways that control MMP9 activation remain undefined. Here, we reported a strong negative correlation of microRNA (miRNA)-181c levels with either MMP9 levels or activation of epidermal growth factor receptor (EGFR) signaling in glioblastoma patients. EGF-induced activation of EGFR in a human glioblastoma line, A-172 cells, increased MMP9 expression through activation of phosphatidylinositol 3-kinase (PI3K)/Akt pathway, without affecting expression of miRNA-181c. On the other hand, overexpression of miRNA-181c in A-172 cells inhibited MMP9 expression by inhibiting Akt phosphorylation, but not phosphorylation of EGFR receptor. Taken together, these findings suggest that EGFR signaling activates downstream PI3K/Akt to increase MMP9 expression in glioblastoma, while phosphorylation of Akt is a control point by miRNA-181c. Our work thus provides new insights into the molecular basis underlying the metastasis of glioblastoma.

Topics: Blotting, Western; Cell Line, Tumor; Chromones; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Matrix Metalloproteinase 9; MicroRNAs; Models, Genetic; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Quinazolines; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Tyrphostins

Accumulating reports suggest that human glioblastoma contains glioma stem-like cells (GSCs) which act as key determinants driving tumor growth, angiogenesis, and contributing to therapeutic resistance. The proliferative signals involved in GSC proliferation and progression remain unclear. Using GSC lines derived from human glioblastoma specimens with different proliferative index and stemness marker expression, we assessed the hypothesis that sphingosine-1-phosphate (S1P) affects the proliferative and stemness properties of GSCs. The results of metabolic studies demonstrated that GSCs rapidly consume newly synthesized ceramide, and export S1P in the extracellular environment, both processes being enhanced in the cells exhibiting high proliferative index and stemness markers. Extracellular S1P levels reached nM concentrations in response to increased extracellular sphingosine. In addition, the presence of EGF and bFGF potentiated the constitutive capacity of GSCs to rapidly secrete newly synthesized S1P, suggesting that cooperation between S1P and these growth factors is of central importance in the maintenance and proliferation of GSCs. We also report for the first time that S1P is able to act as a proliferative and pro-stemness autocrine factor for GSCs, promoting both their cell cycle progression and stemness phenotypic profile. These results suggest for the first time that the GSC population is critically modulated by microenvironmental S1P, this bioactive lipid acting as an autocrine signal to maintain a pro-stemness environment and favoring GSC proliferation, survival and stem properties.

Topics: Animals; Brain Neoplasms; Cell Proliferation; Cells, Cultured; Ceramides; Epidermal Growth Factor; Extracellular Fluid; Fibroblast Growth Factor 2; Fingolimod Hydrochloride; Glioblastoma; Humans; Immunosuppressive Agents; Ki-67 Antigen; Lysophospholipids; Mice; Mice, SCID; Middle Aged; Neoplastic Stem Cells; Propylene Glycols; Sphingolipids; Sphingosine; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Grade IV glioblastoma is characterized by increased kinase activity of epidermal growth factor receptor (EGFR); however, EGFR kinase inhibitors have failed to improve survival in individuals with this cancer because resistance to these drugs often develops. We showed that tumor necrosis factor-α (TNFα) produced in the glioblastoma microenvironment activated atypical protein kinase C (aPKC), thereby producing resistance to EGFR kinase inhibitors. Additionally, we identified that aPKC was required both for paracrine TNFα-dependent activation of the transcription factor nuclear factor κB (NF-κB) and for tumor cell-intrinsic receptor tyrosine kinase signaling. Targeting aPKC decreased tumor growth in mouse models of glioblastoma, including models of EGFR kinase inhibitor-resistant glioblastoma. Furthermore, aPKC abundance and activity were increased in human glioblastoma tumor cells, and high aPKC abundance correlated with poor prognosis. Thus, targeting aPKC might provide an improved molecular approach for glioblastoma therapy.

Topics: Animals; Carcinogenesis; Drug Delivery Systems; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Flow Cytometry; Fluorescent Antibody Technique; Glioblastoma; Humans; Immunoblotting; Immunohistochemistry; Immunoprecipitation; Kaplan-Meier Estimate; Mice; NF-kappa B; Paracrine Communication; Protein Kinase C; Quinazolines; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Tumor Necrosis Factor-alpha

The epidermal growth factor receptor (EGFR) plays pivotal roles in development and is mutated or overexpressed in several cancers. Despite recent advances, the complex allosteric regulation of EGFR remains incompletely understood. Through efforts to understand why the negative cooperativity observed for intact EGFR is lost in studies of its isolated extracellular region (ECR), we uncovered unexpected relationships between ligand binding and receptor dimerization. The two processes appear to compete. Surprisingly, dimerization does not enhance ligand binding (although ligand binding promotes dimerization). We further show that simply forcing EGFR ECRs into preformed dimers without ligand yields ill-defined, heterogeneous structures. Finally, we demonstrate that extracellular EGFR-activating mutations in glioblastoma enhance ligand-binding affinity without directly promoting EGFR dimerization, suggesting that these oncogenic mutations alter the allosteric linkage between dimerization and ligand binding. Our findings have important implications for understanding how EGFR and its relatives are activated by specific ligands and pathological mutations.

Topics: Calorimetry; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Humans; Ligands; Models, Molecular; Mutant Proteins; Mutation; Protein Binding; Protein Multimerization; Protein Structure, Tertiary; Receptors, Fc; Solubility; Thermodynamics

Epidermal growth factor receptor (EGFR) overexpression plays an important oncogenic role in cancer. Regular EGFR protein levels are increased in cancer cells and the receptor then becomes constitutively active. However, downstream signals generated by constitutively activated EGFR are unknown. Here we report that the overexpressed EGFR oscillates between two distinct and mutually exclusive modes of signalling. Constitutive or non-canonical EGFR signalling activates the transcription factor IRF3 leading to expression of IFI27, IFIT1 and TRAIL. Ligand-mediated activation of EGFR switches off IRF3-dependent transcription, activates canonical extracellular signal-regulated kinase (ERK) and Akt signals, and confers sensitivity to chemotherapy and virus-induced cell death. Mechanistically, the distinct downstream signals result from a switch of EGFR-associated proteins. EGFR constitutively complexes with IRF3 and TBK1 leading to TBK1 and IRF3 phosphorylation. Addition of epidermal growth factor dissociates TBK1, IRF3 and EGFR leading to a loss of IRF3 activity, Shc-EGFR association and ERK activation. Finally, we provide evidence for non-canonical EGFR signalling in glioblastoma.

Topics: Adaptor Proteins, Signal Transducing; Antineoplastic Agents; Apoptosis; Brain Neoplasms; Carrier Proteins; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Interferon Regulatory Factor-3; Membrane Proteins; Phosphorylation; Primary Cell Culture; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; RNA-Binding Proteins; Shc Signaling Adaptor Proteins; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand

Receptors of the ErbB family are involved in the development of various cancers, and the inhibition of these receptors represents an attractive therapeutic concept. Upon ligand binding, ErbB receptors become activated as homo- or heterodimers, leading to the activation of downstream signaling cascades that result in the facilitation of cell proliferation and migration. A region of the extracellular part of the receptor, termed the 'dimerization arm', is important for the formation of receptor dimers and represents an attractive target for the design of ErbB inhibitors.. An ErbB1 targeting peptide, termed Herfin-1, was designed based on a model of the tertiary structure of the EGF-EGFR ternary complex. The binding kinetics of this peptide were determined employing surface plasmon resonance analyses. ErbB1-4 expression and phosphorylation in human glioblastoma cell lines U87 and U118 were determined by Western blotting using specific antibodies. Cell proliferation was determined by MTS staining. Cell migration was examined using a Chemotaxis Migration Kit. Neurite outgrowth from primary cerebellar granule neurons was evaluated by fluorescence microscopy and image processing.. The present study shows that Herfin-1 functions as an ErbB1 antagonist. It binds to the extracellular domain of ErbB1 with a KD value of 361 nM. In U87 and U118 cells, both expressing high levels of ErbB1, Herfin-1 inhibits EGF-induced ErbB1 phosphorylation and cell migration. Additionally, Herfin-1 was found to increase neurite outgrowth in cerebellar granule neurons, likely through the inhibition of a sustained weak ErbB1 activation.. Targeting the ErbB1 receptor dimerization interface is a promising strategy to inhibit receptor activation in ErbB1-expressing glioma cells.

Topics: Amino Acid Sequence; Animals; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cells, Cultured; Drug Design; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Humans; Molecular Sequence Data; Neurites; Neurogenesis; Peptides; Phosphorylation; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Rats; Rats, Wistar

Activated EGF receptor (EGFR) signaling plays an instrumental role in glioblastoma (GBM) progression. However, how EGFR activation regulates the tumor microenvironment to promote GBM cell invasion remains to be clarified. Here, we demonstrate that the levels of EGFR activation in tumor cells correlated with the levels of macrophage infiltration in human GBM specimens. This was supported by our observation that EGFR activation enhanced the interaction between macrophages and GBM cells. In addition, EGF treatment induced up-regulation of vascular cell adhesion molecule-1 (VCAM-1) expression in a PKCε- and NF-κB-dependent manner. Depletion of VCAM-1 interrupted the binding of macrophages to GBM cells and inhibited EGF-induced and macrophage-promoted GBM cell invasion. These results demonstrate an instrumental role for EGF-induced up-regulation of VCAM-1 expression in EGFR activation-promoted macrophage-tumor cell interaction and tumor cell invasion and indicate that VCAM-1 is a potential molecular target for improving cancer therapy.

Topics: Cell Communication; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Macrophages; Neoplasm Invasiveness; Neoplasm Proteins; NF-kappa B; Protein Kinase C-epsilon; Tumor Microenvironment; Up-Regulation; Vascular Cell Adhesion Molecule-1

Development of novel patient stratification tools for cancer is a challenge that require advanced molecular screening and a detailed understanding of tumour signalling networks. Here, we apply phospho-specific flow cytometry for signal profiling of primary glioblastoma tumours after preservation of single-cell phosphorylation status as a strategy for evaluation of tumour signalling potential and assessment of rapamycin-mediated mTOR inhibition. The method has already enhanced insight into cancers and disorders of the immune system, and our study demonstrate a great potential to improve the understanding of aberrant signalling in glioblastoma and other solid tumours.

Topics: Antibiotics, Antineoplastic; Base Sequence; Brain Neoplasms; Cell Proliferation; Epidermal Growth Factor; Flow Cytometry; Gene Expression Regulation, Neoplastic; Gene Library; Glioblastoma; Humans; Phosphorylation; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Tumors exhibit complex organization and contain a variety of cell populations. The realization that the regenerative properties of a tumor may be largely confined to a cell subpopulation (cancer stem cell) is driving a new era of anti-cancer research. Cancer stem cells from Glioblastoma Multiforme tumors express markers that are also expressed in non-cancerous neural stem cells, including nestin and Sox2. We previously showed that the transcription factor Hes3 is a marker of neural stem cells, and that its expression is inhibited by JAK activity. Here we show that Hes3 is also expressed in cultures from glioblastoma multiforme which express neural stem cell markers, can differentiate into neurons and glia, and can recapitulate the tumor of origin when transplanted into immunocompromised mice. Similar to observations in neural stem cells, JAK inhibits Hes3 expression. Hes3 RNA interference reduces the number of cultured glioblastoma cells suggesting a novel therapeutic strategy.

Topics: Angiopoietin-2; Animals; Biomarkers; Central Nervous System Neoplasms; DNA-Binding Proteins; Embryonic Stem Cells; Epidermal Growth Factor; Fibroblast Growth Factor 2; Glioblastoma; Janus Kinase 1; Mice; Neoplastic Stem Cells; Phosphorylation; Repressor Proteins; RNA, Small Interfering; STAT3 Transcription Factor; Transcription Factors; Tumor Cells, Cultured

Epidermal growth factor (EGF) and its receptor (EGFR) have been shown to play a significant role in the pathogenesis of glioblastoma. In our study, the EGFR was stimulated with EGF in human U138 glioblastoma cells. We show that the activated mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinases (ERK) 1/2 pathway phosphorylated the E twenty-six (ETS)-like transcription factor 1 (Elk-1) mainly at serine 383 residue. Mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, UO126 and ERK inhibitor II, FR180204 blocked the Elk-1 phosphorylation and activation. The phosphatidylinositide-3-OH kinase (PI3K)/Akt pathway was also involved in the Elk-1 activation. Activation of the Elk-1 led to an increased survival and a proliferative response with the EGF stimulation in the U138 glioblastoma cells. Knocking-down the Elk-1 using an RNA interference technique caused a decrease in survival of the unstimulated U138 glioblastoma cells and also decreased the proliferative response to the EGF stimulation. The Elk-1 transcription factor was important for the survival and proliferation of U138 glioblastoma cells upon the stimulation of EGFR with EGF. The MAPK/ERK1/2 and PI3K/Akt pathways regulated this response via activation of the Elk-1 transcription factor. The Elk-1 may be one of the convergence points for pathways located downstream of EGFR in glioblastoma cells. Utilization of the Elk-1 as a therapeutic target may lead to a novel strategy in treatment of glioblastoma.

Topics: Cell Line, Tumor; Epidermal Growth Factor; ets-Domain Protein Elk-1; Extracellular Signal-Regulated MAP Kinases; Glioblastoma; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Transcription Factors; Transcriptional Activation

Glioblastoma multiforme is a deadly cancer for which current treatment options are limited. The ability of glioblastoma tumor cells to infiltrate the surrounding brain parenchyma critically limits the effectiveness of current treatments. We investigated how microglia, the resident macrophages of the brain, stimulate glioblastoma cell invasion. We first examined the ability of normal microglia from C57Bl/6J mice to stimulate GL261 glioblastoma cell invasion in vitro. We found that microglia stimulate the invasion of GL261 glioblastoma cells by approximately eightfold in an in vitro invasion assay. Pharmacological inhibition of epidermal growth factor receptor (EGFR) strongly inhibited microglia-stimulated invasion. Furthermore, blockade of colony stimulating factor 1 receptor (CSF-1R) signaling using ribonucleic acid (RNA) interference or pharmacological inhibitors completely inhibited microglial enhancement of glioblastoma invasion. GL261 cells were found to constitutively secrete CSF-1, the levels of which were unaffected by epidermal growth factor (EGF) stimulation, EGFR inhibition or coculture with microglia. CSF-1 only stimulated microglia invasion, whereas EGF only stimulated glioblastoma cell migration, demonstrating a synergistic interaction between these two cell types. Finally, using PLX3397 (a CSF-1R inhibitor that can cross the blood-brain barrier) in live animals, we discovered that blockade of CSF-1R signaling in vivo reduced the number of tumor-associated microglia and glioblastoma invasion. These data indicate that glioblastoma and microglia interactions mediated by EGF and CSF-1 can enhance glioblastoma invasion and demonstrate the possibility of inhibiting glioblastoma invasion by targeting glioblastoma-associated microglia via inhibition of the CSF-1R.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Chemotaxis; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Humans; Macrophage Colony-Stimulating Factor; Mice; Mice, Inbred C57BL; Microglia; Neoplasm Invasiveness; Receptor, Macrophage Colony-Stimulating Factor

Despite the high incidence of epidermal growth factor receptor (EGFR) gene amplification and rearrangement in glioblastomas, no suitable cell line exists that preserves these alterations in vitro and is tumorigenic in immunocompromised mice. On the basis of previous observations that glioblastoma cells cultured with serum lose the EGFR amplification rapidly and that EGF can inhibit the growth of EGFR-amplified tumor cells, we hypothesized that serum-free and EGF-free culture conditions could promote maintenance of the EGFR amplification.. Cells from EGFR-amplified glioblastomas were taken into culture using neural stem cell conditions with modifications, including varying oxygen concentrations and omission of routine EGF supplementation.. High-level EGFR amplification was rapidly lost in 5 glioblastoma cultures supplemented with EGF, whereas it was preserved in cultures from the same tumors established without EGF. Cultures from 2 glioblastomas developed into pairs of cell lines, with either stable maintenance or irreversible loss of high-level EGFR amplification in the majority of cells. One EGFR-amplified cell line preserved expression of the receptor variant EGFRvIII. Cell lines with high-level EGFR amplification/EGFRvIII expression formed highly aggressive tumors in nude mice, whereas nonamplified cell lines were either nontumorigenic or grew significantly more slowly. In contrast, nonamplified cell lines proliferated faster in vitro. All cell lines responded to erlotinib, with inhibition of receptor activation and proliferation but partly different effects on downstream signaling and migration.. Isogenic glioblastoma cell lines maintaining stable differences in EGFR/EGFRvIII status can be derived by varying exposure to EGF ligand and reflect the intratumoral genetic heterogeneity.

Topics: Animals; Blotting, Western; Cell Line, Tumor; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Ligands; Mice; Mice, Inbred Strains; Mice, Nude; Neoplasms, Experimental; Neoplastic Stem Cells; Reverse Transcriptase Polymerase Chain Reaction; Transplantation, Heterologous; Tumor Cells, Cultured

Epidermal growth factor receptor (EGFR) signaling is strongly implicated in glioblastoma (GBM) tumorigenesis. However, molecular agents targeting EGFR have demonstrated minimal efficacy in clinical trials, suggesting the existence of GBM resistance mechanisms. GBM cells with stem-like properties (CSCs) are highly efficient at tumor initiation and exhibit therapeutic resistance. In this study, GBMCSC lines showed sphere-forming and tumor initiation capacity after EGF withdrawal from cell culture media, compared with normal neural stem cells that rapidly perished after EGF withdrawal. Compensatory activation of related ERBB family receptors (ERBB2 and ERBB3) was observed in GBM CSCs deprived of EGFR signal (EGF deprivation or cetuximab inhibition), suggesting an intrinsic GBM resistance mechanism for EGFR-targeted therapy. Dual inhibition of EGFR and ERBB2 with lapatinib significantly reduced GBM proliferation in colony formation assays compared to cetuximab-mediated EGFR-specific inhibition. Phosphorylation of downstream ERBB signaling components (AKT, ERK1/2) and GBM CSC proliferation were inhibited by lapatinib. Collectively, these findings show that GBM therapeutic resistance to EGFR inhibitors may be explained by compensatory activation of EGFR-related family members (ERBB2, ERBB3) enabling GBM CSC proliferation, and therefore simultaneous blockade of multiple ERBB family members may be required for more efficacious GBM therapy.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Humans; Mice; Mice, Inbred NOD; Mice, SCID; Neoplastic Stem Cells; Protein Kinase Inhibitors; Signal Transduction; Tumor Burden; Xenograft Model Antitumor Assays

Tumor-specific pyruvate kinase M2 (PKM2) is essential for the Warburg effect. In addition to its well-established role in aerobic glycolysis, PKM2 directly regulates gene transcription. However, the mechanism underlying this nonmetabolic function of PKM2 remains elusive. We show here that PKM2 directly binds to histone H3 and phosphorylates histone H3 at T11 upon EGF receptor activation. This phosphorylation is required for the dissociation of HDAC3 from the CCND1 and MYC promoter regions and subsequent acetylation of histone H3 at K9. PKM2-dependent histone H3 modifications are instrumental in EGF-induced expression of cyclin D1 and c-Myc, tumor cell proliferation, cell-cycle progression, and brain tumorigenesis. In addition, levels of histone H3 T11 phosphorylation correlate with nuclear PKM2 expression levels, glioma malignancy grades, and prognosis. These findings highlight the role of PKM2 as a protein kinase in its nonmetabolic functions of histone modification, which is essential for its epigenetic regulation of gene expression and tumorigenesis.

Topics: Animals; Astrocytoma; Carrier Proteins; Cell Line; Cell Line, Tumor; Cell Transformation, Neoplastic; Cyclin D1; Epidermal Growth Factor; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Histones; Humans; Membrane Proteins; Mice; Mice, Nude; Neoplasm Transplantation; Proto-Oncogene Proteins c-myc; Thyroid Hormone-Binding Proteins; Thyroid Hormones; Transcription, Genetic; Transplantation, Heterologous

Glioblastoma is one of the most aggressive tumors with poor prognosis. Although various studies have been performed so far, there are not effective treatments for patients with glioblastoma.. In order to systematically elucidate the aberrant signaling machinery activated in this malignant brain tumor, we investigated phosphoproteome dynamics of glioblastoma initiating cells using high-resolution nanoflow LC-MS/MS system in combination with SILAC technology. Through phosphopeptide enrichment by titanium dioxide beads, a total of 6,073 phosphopeptides from 2,282 phosphorylated proteins were identified based on the two peptide fragmentation methodologies of collision induced dissociation and higher-energy C-trap dissociation. The SILAC-based quantification described 516 up-regulated and 275 down-regulated phosphorylation sites upon epidermal growth factor stimulation, including the comprehensive status of the phosphorylation sites on stem cell markers such as nestin. Very intriguingly, our in-depth phosphoproteome analysis led to identification of novel phosphorylated molecules encoded by the undefined sequence regions of the human transcripts, one of which was regulated upon external stimulation in human glioblastoma initiating cells.. Our result unveils an expanded diversity of the regulatory phosphoproteome defined by the human transcriptome.

Topics: Amino Acid Sequence; Amino Acids; Blotting, Western; Brain Neoplasms; Cell Line, Tumor; Chromatography, Liquid; Epidermal Growth Factor; Fibroblast Growth Factors; Glioblastoma; Humans; Isotope Labeling; Mass Spectrometry; Molecular Sequence Data; Neoplastic Stem Cells; Phosphopeptides; Phosphoproteins; Proteome; Proteomics; Sequence Homology, Amino Acid; Signal Transduction; Transcriptome

The invasion of glioblastoma cells into regions of the normal brain is a critical factor that limits current therapies for malignant astrocytomas. Previous work has identified roles for the Rho family guanine nucleotide exchange factors Trio and Vav3 in glioblastoma invasion. Both Trio and Vav3 act on the small GTPase RhoG. We therefore examined the role of RhoG in the invasive behavior of glioblastoma cells.. We found that siRNA-mediated depletion of RhoG strongly inhibits invasion of glioblastoma cells through brain slices ex vivo. In addition, depletion of RhoG has a marginal effect on glioblastoma cell proliferation, but significantly inhibits glioblastoma cell survival in colony formation assays. We also observed that RhoG is activated by both HGF and EGF, two factors that are thought to be clinically relevant drivers of glioblastoma invasive behavior, and that RhoG is overexpressed in human glioblastoma tumors versus non-neoplastic brain. In search of a mechanism for the contribution of RhoG to the malignant behavior of glioblastoma cells, we found that depletion of RhoG strongly inhibits activation of the Rac1 GTPase by both HGF and EGF. In line with this observation, we also show that RhoG contributes to the formation of lamellipodia and invadopodia, two functions that have been shown to be Rac1-dependent.. Our functional analysis of RhoG in the context of glioblastoma revealed a critical role for RhoG in tumor cell invasion and survival. These results suggest that targeting RhoG-mediated signaling presents a novel avenue for glioblastoma therapy.

Topics: Animals; Brain Neoplasms; Cell Growth Processes; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Humans; Mice; Neoplasm Invasiveness; Neuropeptides; Pseudopodia; Putamen; rac GTP-Binding Proteins; rac1 GTP-Binding Protein; rho GTP-Binding Proteins; RNA, Small Interfering

Many types of human tumor cells have overexpressed pyruvate kinase M2 (PKM2). However, the mechanism underlying this increased PKM2 expression remains to be defined. We demonstrate here that EGFR activation induces PLCγ1-dependent PKCε monoubiquitylation at Lys321 mediated by RINCK1 ubiquitin ligase. Monoubiquitylated PKCε interacts with a ubiquitin-binding domain in NEMO zinc finger and recruits the cytosolic IKK complex to the plasma membrane, where PKCε phosphorylates IKKβ at Ser177 and activates IKKβ. Activated RelA interacts with HIF1α, which is required for RelA to bind the PKM promoter. PKCε- and NF-κB-dependent PKM2 upregulation is required for EGFR-promoted glycolysis and tumorigenesis. In addition, PKM2 expression correlates with EGFR and IKKβ activity in human glioblastoma specimens and with grade of glioma malignancy. These findings highlight the distinct regulation of NF-κB by EGF, in contrast to TNF-α, and the importance of the metabolic cooperation between the EGFR and NF-κB pathways in PKM2 upregulation and tumorigenesis.

Topics: Animals; Brain Neoplasms; Carrier Proteins; Cell Line, Tumor; Cell Transformation, Neoplastic; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Genes, Reporter; Glioblastoma; Glucose; Glycolysis; HEK293 Cells; Heterogeneous-Nuclear Ribonucleoproteins; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; I-kappa B Kinase; Lactic Acid; Membrane Proteins; Mice; Mice, Nude; Mutagenesis, Site-Directed; Mutation; Neoplasm Grading; Neoplasm Transplantation; NF-kappa B; Phospholipase C gamma; Phosphorylation; Polypyrimidine Tract-Binding Protein; Prognosis; Promoter Regions, Genetic; Protein Kinase C-epsilon; RNA Interference; Serine; Signal Transduction; Thyroid Hormone-Binding Proteins; Thyroid Hormones; Transcription Factor RelA; Transfection; Ubiquitination; Up-Regulation

Alternative exon usage (AEU) is an important component of gene regulation. Exon expression platforms allow the detection of associations between AEU and phenotypes such as cancer. Numerous studies have identified associations between gene expression and the brain cancer glioblastoma multiforme (GBM). The few consistent gene expression biomarkers of GBM that have been reported may be due to the limited consideration of AEU and the analytical approaches used. The objectives of this study were to develop a model that accounts for the variations in expression present between the exons within a gene and to identify AEU biomarkers of GBM survival.. The expression of exons corresponding to 25,403 genes was related to the survival of 250 individuals diagnosed with GBM in a training data set. Genes exhibiting AEU in the training data set were confirmed in an independent validation data set of 78 patients. A hierarchical mixed model that allows the consideration of covariation between exons within a gene and of the effect of the epidemiological characteristics of the patients was developed to identify associations between exon expression and patient survival. This general model describes all three possible scenarios: multi-exon genes with and without AEU, and single-exon genes.. AEU associated with GBM survival was identified on 2477 genes (P-value < 5.0E-04 or FDR-adjusted P-value < 0.05). G-protein coupled receptor 98 (Gpr98) and epidermal growth factor (Egf) were among the genes exhibiting AEU with 30 and 9 exons associated with GBM survival, respectively. Pathways enriched among the AEU genes included focal adhesion, ECM-receptor interaction, ABC transporters and pathways in cancer. In addition, 24 multi-exon genes without AEU and 8 single-exon genes were associated with GBM survival (FDR-adjusted P-value < 0.05).. The inferred patterns of AEU were consistent with in silico AS models. The hierarchical model used offered a flexible and simple way to interpret and identify associations between survival that accommodates multi-exon genes with or without AEU and single exon genes. Our results indicate that differential expression of AEU could be used as biomarker for GBM and potentially other cancers.

Topics: Alternative Splicing; Epidermal Growth Factor; Exons; Female; Genome, Human; Glioblastoma; Humans; Male; Models, Genetic; Odds Ratio; Oligonucleotide Array Sequence Analysis; Receptors, G-Protein-Coupled; Survival Analysis

Rapidly growing tumors require efficient means to allow them to adapt to fluctuating microenvironments consisting of hypoxia, nutrient deprivation, and acidosis. The unfolded protein response (UPR) represents a defense mechanism allowing cells to respond to these adverse conditions. The chaperone protein GRP78 serves as a master UPR regulator that is aberrantly expressed in a variety of cancers, including glioma. Therefore, cancer cells may be particularly reliant upon the adaptive mechanisms offered by the UPR and targeting GRP78 may represent a unique therapeutic strategy. Here we report that diffuse expression of GRP78 protein is present in Grade III-IV, but not Grade I-II glioma. To determine the role GRP78 plays in glioblastoma tumorigenesis, we explored the anti-tumor activity of the novel fusion protein EGF-SubA, which combines EGF with the cytotoxin SubA that has been recently shown to selectively cleave GRP78. EGF-SubA demonstrated potent tumor-specific proteolytic activity and cytotoxicity in glioblastoma lines and potentiated the anti-tumor activity of both temozolomide and ionizing radiation. To determine if the tumor microenvironment influences EGF-SubA activity, we maintained cells in acidic conditions that led to both UPR activation and increased EGF-SubA induced cytotoxicity. EGF-SubA was well tolerated in mice and led to a significant tumor growth delay in a glioma xenograft mouse model. The UPR is emerging as an important adaptive pathway contributing to glioma tumorigenesis. Targeting its primary mediator, the chaperone protein GRP78, through specific, proteolytic cleavage with the immunotoxin EGF-SubA represents a novel and promising multi-targeted approach to cancer therapy.

Topics: Animals; Cell Line, Tumor; Endoplasmic Reticulum Chaperone BiP; Epidermal Growth Factor; Glioblastoma; Heat-Shock Proteins; Humans; Immunoblotting; Mice; Reverse Transcriptase Polymerase Chain Reaction; Subtilisins; Tissue Array Analysis; Unfolded Protein Response

To develop a novel polyethylenimine (PEI)-based polymeric carrier for tumor-targeted delivery of cytotoxic double-stranded RNA polyinosinic:polycytidylic acid, poly(I:C). The novel carrier should be chemically less complex but at least as effective as a previously developed tetra-conjugate containing epidermal growth factor (EGF) as targeting ligand, polyethylene glycol (PEG) as shielding spacer, 25 kDa branched PEI as RNA binding and endosomal buffering agent, and melittin as endosomal escape agent.. Novel conjugates were designed employing a simplified synthetic strategy based on 22 kDa linear polyethylenimine (LPEI), PEG spacers, and recombinant EGF. The efficacy of various conjugates (different PEG spacers, with and without targeting EGF) in poly(I:C)-mediated cell killing was evaluated in vitro using two human U87MG glioma cell lines. The most effective polyplex was tested for in vivo activity in A431 tumor xenografts.. Targeting conjugate LPEI-PEG2 kDa-EGF was found as most effective in poly(I:C)-triggered killing of tumor cells in vitro. The efficacy correlated with glioma cell EGFR levels. Repeated intravenous administration of poly(I:C) polypexes strongly retarded growth of A431 human tumor xenograft in mice.. The optimized LPEI-PEG2 kDa-EGF conjugate displays reduced chemical complexity and efficient poly(I:C)-mediated killing of EGFR overexpressing tumors in vitro and in vivo.

Topics: Animals; Antineoplastic Agents; Cell Culture Techniques; Cell Line, Tumor; Cell Survival; Drug Carriers; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Humans; Mice; Mice, Nude; Poly I-C; Polyethylene Glycols; Polyethyleneimine; Xenograft Model Antitumor Assays

We have previously shown that high expression levels of the lipid kinase sphingosine kinase-1 (SphK1) correlate with poor survival of glioblastoma (GBM) patients. In this study we examined the regulation of SphK1 expression by epidermal growth factor receptor (EGFR) signaling in GBM cells. As the EGFR gene is often overexpressed and mutated in GBM, and EGFR has been shown to regulate SphK1 in some cell types, we examined the effect of EGF signaling and the constitutively active EGFRvIII mutant on SphK1 in GBM cells. Treatment of glioma cell lines with EGF led to increased expression and activity of SphK1. Expression of EGFRvIII in glioma cells also activated and induced SphK1. In addition, siRNA to SphK1 partially inhibited EGFRvIII-induced growth and survival of glioma cells as well as ERK MAP kinase activation. To further evaluate the connection between EGFR and SphK1 in GBM we examined primary neurosphere cells isolated from fresh human GBM tissue. The GBM-derived neurosphere cell line GBM9, which forms GBM-like tumors intracranially in nude mice, maintained expression of EGFRvIII in culture and had high levels of SphK1 activity. EGFR inhibitors modestly decreased SphK1 activity and proliferation of GBM9 cells. More extensive blockage of SphK1 activity by a SphK inhibitor, potently blocked cell proliferation and induced apoptotic cell death of GBM9 cells. Thus, SphK1 activity is necessary for survival of GBM-derived neurosphere cells, and EGFRvIII partially utilizes SphK1 to further enhance cell proliferation.

Topics: Animals; Annexin A5; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Disease Models, Animal; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Mice; Mice, Nude; Mutation; Phosphotransferases (Alcohol Group Acceptor); RNA, Small Interfering; Signal Transduction; Time Factors

Using an orthotopic intracerebral model from our established HM55-BGIV-101 tumor line, we investigated the antitumor effect on the angiogenesis and growth of human glioblastoma after treatment with monoclonal antibody DC101 against the vascular endothelial growth factor receptor-2 and monoclonal antibody C225 against the epidermal growth factor receptor. Nude mice bearing intracerebral glioblastoma xenografts were treated intraperitoneally with DC101 and C225 either alone or in combination. Histopathological analysis of solid tumor volume, satellite tumor number, microvessel density, tumor cell proliferation, and apoptosis was performed. In the DC101-treated group, solid tumor volume and microvessel density were reduced by 59.7 and 64%, respectively; tumor cell proliferative activity was reduced by 53.2% and the apoptotic index (AI) was increased by 66.7%; satellite tumor number was enhanced by 84.4%. C225 alone reduced satellite tumor number by 43.3%, but had no effect on solid tumor volume, microvessel density, tumor cell proliferation, and apoptosis. C225 combined with DC101 not only reduced solid tumor volume, microvessel density, tumor cell proliferative activity, and increased AI, but also reduced satellite tumor number. Inhibition of angiogenesis achieved by DC101 can cause increased tumor cell invasiveness. In our studies this increased tumor cell invasiveness was inhibited simultaneously by C225, which provides a theoretical basis for treatment of glioblastoma by the method of combining drugs with different pharmacological activity.

Topics: Analysis of Variance; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antigens, CD34; Brain Neoplasms; Cell Proliferation; Cetuximab; Disease Models, Animal; Epidermal Growth Factor; Glioblastoma; Humans; In Situ Nick-End Labeling; Male; Mice; Middle Aged; Neoplasm Transplantation; Protein Precursors; Survival Analysis; Time Factors; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

Platelet derived growth factor (PDGF) regulates gene transcription by binding to specific receptors. PDGF plays a critical role in oncogenesis in brain and other tumors, regulates angiogenesis, and remodels the stroma in physiologic conditions. Here, we show by using microRNA (miR) arrays that PDGFs regulate the expression and function of miRs in glioblastoma and ovarian cancer cells. The two PDGF ligands AA and BB affect expression of several miRs in ligand-specific manner; the most robust changes consisting of let-7d repression by PDGF-AA and miR-146b induction by PDGF-BB. Induction of miR-146b by PDGF-BB is modulated via MAPK-dependent induction of c-fos. We demonstrate that PDGF regulates expression of some of its known targets (e.g. cyclin D1) through miR alterations and identify the epidermal growth factor receptor (EGFR) as a new PDGF-BB target. We show that its expression and function are repressed by PDGF-induced miR-146b and that mir-146b and EGFR correlate inversely in human glioblastomas. We propose that PDGF-regulated gene transcription involves alterations in non-coding RNAs and provide evidence for a miR-dependent feedback mechanism balancing growth factor receptor signaling in cancer cells.

Topics: Becaplermin; Cell Line; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; MicroRNAs; Ovarian Neoplasms; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Signal Transduction

The authors of this study aimed to genetically design a bispecific targeted toxin that would simultaneously target overexpressed markers on glioma as well as the tumor vasculature, to mutate certain amino acids to reduce the immunogenicity of this new drug, and to determine whether the drug was able to effectively reduce aggressive human brain tumors in a rat xenograft model via a novel hollow fiber (HF) catheter delivery system.. A new bispecific ligand-directed toxin (BLT) was created in which 2 human cytokines-epidermal growth factor ([EGF], targeting overexpressed EGF receptor) and amino acid terminal fragment ([ATF], targeting urokinase plasminogen activator receptor)-were cloned onto the same single-chain molecule with truncated Pseudomonas exotoxin with a terminal lysyl-aspartyl-glutamyl-leucine (KDEL) sequence. Site-specific mutagenesis was used to mutate amino acids in 7 key epitopic toxin regions that dictate the B cell generation of neutralizing antitoxin antibodies to deimmunize the drug, now called "EGFATFKDEL 7mut." Bioassays were used to determine whether mutation reduced the drug's potency, and enzyme-linked immunosorbent assay studies were performed to determine whether antitoxin antibodies were decreased. Aggressive brain tumors were intracranially established in nude rats by using human U87 glioma genetically marked with a firefly luciferase reporter gene (U87-luc), and the rats were stereotactically treated with 2 intracranial injections of deimmunized EGFATFKDEL via convection-enhanced delivery (CED). Drug was administered through a novel HF catheter to reduce drug backflow upon delivery.. In vitro, EGFATFKDEL 7mut selectively killed the human glioblastoma cell line U87-luc as well as cultured human endothelial cells in the form of the human umbilical vein endothelial cells. Deimmunization did not reduce drug activity. In vivo, when rats with brain tumors were intracranially treated with drug via CED and a novel HF catheter to reduce backflow, there were significant tumor reductions in 2 experiments (p < 0.01). Some rats survived with a tumor-free status until 130 days post-tumor inoculation. An irrelevant BLT control did not protect establishing specificity. The maximal tolerated dose of EGFATFKDEL 7mut was established at 2 μg/injection or 8.0 μg/kg, and data indicated that this dose was nontoxic. Antitoxin antibodies were reduced by at least 90%.. First, data indicated that the BLT framework is effective for simultaneously targeting glioma and its neovasculature. Second, in the rodent CED studies, newly developed HF catheters that limit backflow are effective for drug delivery. Third, by mutating critical amino acids, the authors reduced the threat of the interference of neutralizing antibodies that are generated against the drug. The authors' experiments addressed some of the most urgent limitations in the targeted toxin field.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Drug Design; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Glioblastoma; Humans; Immunotoxins; Mutagenesis, Site-Directed; Neoplasm Transplantation; Neovascularization, Pathologic; Rats; Rats, Nude; Receptors, Urokinase Plasminogen Activator; Xenograft Model Antitumor Assays

Glioblastoma (GBM) is a highly aggressive malignant primary brain tumor, characterized by rapid growth, diffuse infiltration of cells into both adjacent and remote brain regions, and a generalized resistance to currently available treatment modalities. Recent reports in the literature suggest that Signal Transducers and Activators of Transcription (STATs) play important roles in the regulation of GBM pathophysiology.. STAT6 protein expression was analyzed by Western blotting in GBM cell lines and by immunohistochemistry in a tissue microarray (TMA) of glioma patient tissues. We utilized shRNA against STAT6 to investigate the effects of prolonged STAT6 depletion on the growth and invasion of two STAT6-positive GBM cell lines. Cell proliferation was assessed by measuring (3)H-Thymidine uptake over time. Invasion was measured using an in vitro transwell assay in which cells invade through a type IV collagen matrix toward a chemoattractant (Fetal Bovine Serum). Cells were then stained and counted. Kaplan-Meyer survival curves were generated to show the correlation between STAT6 gene expression and patient survival in 343 glioma patients and in a subset of patients with only GBM. Gene expression microarray and clinical data were acquired from the Rembrandt 1 public data depository (https://caintegrator.nci.nih.gov/rembrandt/). Lastly, a genome-wide expression microarray analysis was performed to compare gene expression in wild-type GBM cells to expression in stable STAT6 knockdown clones.. STAT6 was expressed in 2 GBM cell lines, U-1242MG and U-87MG, and in normal astrocytes (NHA) but not in the U-251MG GBM cell line. In our TMA study, STAT6 immunostaining was visible in the majority of astrocytomas of all grades (I-IV) but not in normal brain tissue. In positive cells, STAT6 was localized exclusively in the nuclei over 95% of the time. STAT6-deficient GBM cells showed a reduction in (3)H-Thymidine uptake compared to the wild-type. There was some variation among the different shRNA- silenced clones, but all had a reduction in (3)H-Thymidine uptake ranging from 35%- 70% in U-1242MG and 40- 50% in U-87MG cells. Additionally, STAT6- depleted cells were less invasive than controls in our in vitro transmembrane invasion assay. Invasiveness was decreased by 25-40% and 30-75% in U-1242MG and U-87MG cells, respectively. The microarray analysis identified matrix metalloproteinase 1 (MMP-1) and urokinase Plasminogen activator (uPA) as potential STA6 target genes involved in the promotion of GBM cell invasion. In a Kaplan-Meier survival curve based on Rembrandt 1 gene expression microarray and clinical data, there was a significant difference in survival (P < 0.05) between glioma patients with up- and down-regulated STAT6. Decreased STAT6 expression correlated with longer survival times. In two subsets of patients with either grade IV tumors (GBM) or Grade II/III astrocytomas, there was a similar trend that however did not reach statistical significance.. Taken together, these findings suggest a role for STAT6 in enhancing cell proliferation and invasion in GBM, which may explain why up-regulation of STAT6 correlates with shorter survival times in glioma patients. This report thus identifies STAT6 as a new and potentially promising therapeutic target.

Topics: Astrocytoma; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Databases, Factual; Epidermal Growth Factor; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Silencing; Glioblastoma; Glioma; Humans; Neoplasm Invasiveness; Phosphorylation; RNA, Small Interfering; STAT6 Transcription Factor; Survival Analysis; Tyrosine

Glioblastoma (GBM) is among the most lethal of all cancers. GBM consist of a heterogeneous population of tumor cells among which a tumor-initiating and treatment-resistant subpopulation, here termed GBM stem cells, have been identified as primary therapeutic targets. Here, we describe a high-throughput small molecule screening approach that enables the identification and characterization of chemical compounds that are effective against GBM stem cells. The paradigm uses a tissue culture model to enrich for GBM stem cells derived from human GBM resections and combines a phenotype-based screen with gene target-specific screens for compound identification. We used 31,624 small molecules from 7 chemical libraries that we characterized and ranked based on their effect on a panel of GBM stem cell-enriched cultures and their effect on the expression of a module of genes whose expression negatively correlates with clinical outcome: MELK, ASPM, TOP2A, and FOXM1b. Of the 11 compounds meeting criteria for exerting differential effects across cell types used, 4 compounds showed selectivity by inhibiting multiple GBM stem cells-enriched cultures compared with nonenriched cultures: emetine, n-arachidonoyl dopamine, n-oleoyldopamine (OLDA), and n-palmitoyl dopamine. ChemBridge compounds #5560509 and #5256360 inhibited the expression of the 4 mitotic module genes. OLDA, emetine, and compounds #5560509 and #5256360 were chosen for more detailed study and inhibited GBM stem cells in self-renewal assays in vitro and in a xenograft model in vivo. These studies show that our screening strategy provides potential candidates and a blueprint for lead compound identification in larger scale screens or screens involving other cancer types.

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; Cell Growth Processes; Cell Line, Tumor; Cell Transformation, Neoplastic; Culture Media, Serum-Free; Emetine; Epidermal Growth Factor; Fibroblast Growth Factors; Gene Expression; Glioblastoma; High-Throughput Screening Assays; Humans; Mice; Mice, Inbred NOD; Mice, SCID; Neoplastic Stem Cells; Xenograft Model Antitumor Assays

Few prognostic factors have been associated with glioblastoma survival. We analyzed a complete tagging of the epidermal growth factor (EGF) and EGF receptor (EGFR) gene polymorphisms as potential prognostic factors. Thirty tagging single-nucleotide polymorphisms (SNPs) in EGF and 89 tagging SNPs in EGFR were analyzed for association with survival in 176 glioblastoma cases. Validation analyses were performed for 4 SNPs in a set of 638 glioblastoma patients recruited at The University of Texas M. D. Anderson Cancer Center (MDACC). Three hundred and seventy-four glioblastoma patients aged 50 years or older at diagnosis were subanalyzed to enrich for de novo arising glioblastoma. We found 7 SNPs in haplotype 4 in EGF that were associated with prognosis in glioblastoma patients. In EGFR, 4 of 89 SNPs were significantly associated with prognosis but judged as false positives. Four of the significantly associated EGF polymorphisms in haplotype block 4 were validated in a set from MDACC; however, none of the associations were clearly replicated. rs379644 had a hazard ratio (HR) of 1.19 (0.94-1.51) in the whole population with 18.6 months survival in the risk genotype compared with 24.5 in the reference category. As the median age differed slightly between the 2 study sets, the MDACC cases aged 50 or older at diagnosis were analyzed separately (rs379644, HR 1.32 [0.99-1.78]), which is marginally significant and partially validates our findings. This study is, to our knowledge, the first to perform a comprehensive tagging of the EGF and EGFR genes, and the data give some support that EGF polymorphisms might be associated with poor prognosis. Further confirmation in independent data sets of prospective studies is necessary to establish EGF as prognostic risk factor.

Topics: Adult; Aged; Brain Neoplasms; Epidermal Growth Factor; ErbB Receptors; Female; Genotype; Glioblastoma; Humans; Male; Middle Aged; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Prognosis; Risk Factors; Young Adult

The activation of ion channels is crucial during cell movement, including glioblastoma cell invasion in the brain parenchyma. In this context, we describe for the first time the contribution of intermediate conductance Ca(2+)-activated K (IK(Ca)) channel activity in the chemotactic response of human glioblastoma cell lines, primary cultures, and freshly dissociated tissues to CXC chemokine ligand 12 (CXCL12), a chemokine whose expression in glioblastoma has been correlated with its invasive capacity. We show that blockade of the IK(Ca) channel with its specific inhibitor 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) or IK(Ca) channel silencing by short hairpin RNA (shRNA) completely abolished CXCL12-induced cell migration. We further demonstrate that this is not a general mechanism in glioblastoma cell migration since epidermal growth factor (EGF), which also activates IK(Ca) channels in the glioblastoma-derived cell line GL15, stimulate cell chemotaxis even if the IK(Ca) channels have been blocked or silenced. Furthermore, we demonstrate that both CXCL12 and EGF induce Ca(2+) mobilization and IK(Ca) channel activation but only CXCL12 induces a long-term upregulation of the IK(Ca) channel activity. Furthermore, the Ca(2+)-chelating agent BAPTA-AM abolished the CXCL12-induced, but not the EGF-induced, glioblastoma cell chemotaxis. In addition, we demonstrate that the extracellular signal-regulated kinase (ERK)1/2 pathway is only partially implicated in the modulation of CXCL12-induced glioblastoma cell movement, whereas the phosphoinositol-3 kinase (PI3K) pathway is not involved. In contrast, EGF-induced glioblastoma migration requires both ERK1/2 and PI3K activity. All together these findings suggest that the efficacy of glioblastoma invasiveness might be related to an array of nonoverlapping mechanisms activated by different chemotactic agents.

Topics: Brain Neoplasms; Calcium Signaling; Cell Line, Tumor; Chelating Agents; Chemokine CXCL12; Chemotaxis; Epidermal Growth Factor; Glioblastoma; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Membrane Potentials; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Potassium Channel Blockers; Protein Kinase Inhibitors; Receptors, CXCR4; Recombinant Proteins; RNA Interference; Tumor Cells, Cultured

The lack of an intracranial human glioma model that recapitulates the extensive invasive and hypervascular features of glioblastoma (GBM) is a major hurdle for testing novel therapeutic approaches against GBM and studying the mechanism of GBM invasive growth. We characterized a high matrix metalloproteinase-9 (MMP-9) expressing U1242 MG intracranial xenograft mouse model that exhibited extensive individual cells and cell clusters in a perivascular and subpial cellular infiltrative pattern, geographic necrosis and infiltrating tumor-induced vascular proliferation closely resembling the human GBM phenotype. MMP-9 silencing cells with short hairpin RNA dramatically blocked the cellular infiltrative pattern, hypervascularity, and cell proliferation in vivo, and decreased cell invasion, colony formation, and cell motility in vitro, indicating that a high level of MMP-9 plays an essential role in extensive infiltration and hypervascularity in the xenograft model. Moreover, epidermal growth factor (EGF) failed to stimulate MMP-9 expression, cell invasion, and colony formation in MMP-9-silenced clones. An EGF receptor (EGFR) kinase inhibitor, a RasN17 dominant-negative construct, MEK and PI3K inhibitors significantly blocked EGF/EGFR-stimulated MMP-9, cell invasion, and colony formation in U1242 MG cells, suggesting that MMP-9 is involved in EGFR/Ras/MEK and PI3K/AKT signaling pathway-mediated cell invasion and anchorage-independent growth in U1242 MG cells. Our data indicate that the U1242 MG xenograft model is valuable for studying GBM extensive invasion and angiogenesis as well as testing anti-invasive and anti-angiogenic therapeutic approaches.

Topics: Animals; Astrocytes; Brain Neoplasms; Cell Movement; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Humans; Male; Matrix Metalloproteinase 9; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Invasiveness; Neoplasm Transplantation; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; ras Proteins; RNA, Small Interfering; Signal Transduction; Transplantation, Heterologous

One of the major pathophysiological features of malignant astrocytomas is their ability to infiltrate surrounding brain tissue. The epidermal growth factor receptor (EGFR) and proteases are known to be overexpressed in glioblastomas (GBMs), but the interaction between the activation of the EGFR and urokinase plasminogen activator (uPA) in promoting astrocytic tumor invasion has not been fully elucidated. Here, we characterized the signal transduction pathway(s) by which EGF regulates uPA expression and promotes astrocytoma invasion. We show that EGFR activation and constitutively active EGFR vIII in GBM cell lines upregulate uPA expression. Small-molecule inhibitors of mitogen-activated protein kinase, tyrosine kinase, and small interfering RNA targeting c-Src blocked uPA upregulation. Similarly, mutations in the activator protein 1 binding site of the uPA promoter reduced EGF-induced increases in uPA promoter activity. Treatment of GBM cells with EGF increased in vitro cell invasion, and the invasive phenotype was attenuated by gene silencing of uPA using small interfering RNA and short hairpin RNA. In addition, uPA knockdown clones formed smaller well-circumscribed tumors than nontarget U1242 control cells in a xenograft GBM mouse model in vivo. In summary, these results suggest that c-Src, mitogen-activated protein kinase, and a composite activator protein 1 on the uPA promoter are responsible for EGF-induced uPA expression and GBM invasion.

Topics: Animals; Astrocytes; Blotting, Northern; Blotting, Western; Cell Line; Cell Movement; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Humans; Image Processing, Computer-Assisted; Magnetic Resonance Imaging; MAP Kinase Signaling System; Mice; Promoter Regions, Genetic; RNA, Messenger; RNA, Small Interfering; Time Factors; Transcription Factor AP-1; Transfection; Transplantation, Heterologous; Urokinase-Type Plasminogen Activator

Macroautophagy (hereafter referred to as autophagy) can increase or decrease the amount of cell death in response to various stimuli. To test whether autophagy also controls the characteristics associated with dying cells, we studied tumor cell killing by epidermal growth factor receptor-targeted diphtheria toxin (DT-EGF). DT-EGF kills epithelial and glioblastoma tumor cells with similar efficiency but by different mechanisms that depend on whether the cells activate autophagy when treated with the drug. Dying cells in which autophagy is induced selectively release the immune modulator high-mobility group B1 (HMGB1) without causing lysis of the cell membrane and classical necrosis. Conversely, cells that are killed by DT-EGF where autophagy is blocked, activate caspases but retain HMGB1. These data suggest that it may be feasible to manipulate the immunogenicity of dying cells by increasing or decreasing autophagy.

Topics: Autophagy; Cell Line, Tumor; Diphtheria Toxin; Epidermal Growth Factor; Glioblastoma; HMGB1 Protein; Humans; Immunologic Factors; Neoplasm Proteins; Neoplasms, Glandular and Epithelial; Recombinant Fusion Proteins

Glioblastoma stem cells are able to reform original glioblastoma and express the neural stem cell marker CD133 and Nestin. They can self-renew and proliferate in tumor sphere medium containing EGF, bFGF and LIF that is known to be permissive for stem cell proliferation. In this study, we found that neurosphere-like colonies appeared after the human primary glioblastoma cells had been switched into pure DMEM/F12 medium. We investigated whether tumor spheres formed in pure DMEM/F12 medium possess the characteristics of glioblastoma stem cells. We identified that the tumor sphere cells were cancer stem cells of glioblastoma and they can self-renew and proliferate in pure DMEM/F12 medium. Glioblastoma cells can secrete several factors that result in autocrine motility signaling and stimulate glioma invasion. We hypothesized that an essential autocrine signal promotes the self-renewal and proliferation of human glioblastoma stem cells in pure DMEM/F12 medium. Then, expression of EGF and bFGF in glioblastoma stem cells were analyzed. Both the mRNA and protein of EGF and bFGF were detected in three human glioblastoma stem cells. Our findings suggest that autocrine of EGF and bFGF may sustain the self-renewal of glioblastoma stem cells.

Topics: Animals; Autocrine Communication; Blotting, Western; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Culture Media; Epidermal Growth Factor; Fibroblast Growth Factor 2; Flow Cytometry; Glioblastoma; Humans; Immunohistochemistry; Mice; Mice, Nude; Neoplastic Stem Cells; Reverse Transcriptase Polymerase Chain Reaction; Spheroids, Cellular

A growing body of evidence suggests that glioma stem-like cells are more representative of their parent tumours when cultured under defined serum-free conditions with the mitogens epidermal growth factor (EGF) and fibroblast growth factor (FGF). However, culturing these cells as free-floating spheroids can result in difficulty in efficiently deriving and propagating cell lines. We have combined neurosphere and monolayer culture techniques to improve the efficiency with which cells can be derived from clinical tumour samples under defined serum-free conditions. We have applied our protocol to consecutive samples of glioblastoma to show that they can form experimental tumours that recapitulate many of the histological features of the parent tumour. We go on to show that the tumour initiating cells also retain the cytogenetic abnormalities of the parent tumour. Finally we examined the cell lines for expression of markers associated with neural stem cells. Our results confirm the expression of transcription factors associated with neural patterning and specification including Sox2, Olig2, Pax6 and Nkx2.2. We went on to establish that these factors were also expressed in the parent tumour indicating that their expression was not a function of our culture conditions. The Cambridge Protocol is an efficient method of deriving stem-like tumour initiating cells from glioblastoma. Improving the efficiency of derivation will facilitate the improvement of in vitro and in vivo model systems to study disease mechanisms, screen drugs and develop novel therapeutic approaches in the future.

Topics: Animals; Brain Neoplasms; Cell Culture Techniques; Cell Line, Tumor; Culture Media, Conditioned; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Glioblastoma; Homeobox Protein Nkx-2.2; Homeodomain Proteins; Humans; Mice; Mice, SCID; Neoplasm Transplantation; Nerve Tissue Proteins; Nuclear Proteins; Transcription Factors; Tumor Cells, Cultured

Hypoxia and necrosis are fundamental features of glioblastoma (GBM) and their emergence is critical for the rapid biological progression of this fatal tumor; yet, underlying mechanisms are poorly understood. We have suggested that vaso-occlusion following intravascular thrombosis could initiate or propagate hypoxia and necrosis in GBM. Tissue factor (TF), the main cellular initiator of coagulation, is overexpressed in GBMs and likely favors a thrombotic microenvironment. Epidermal growth factor receptor (EGFR) amplification and PTEN loss are two common genetic alterations seen in GBM but not in lower-grade astrocytomas that could be responsible for TF up-regulation. The most frequent EGFR mutation in GBM involves deletion of exons 2 to 7, resulting in the expression of a constitutively active receptor, EGFRvIII. Here, we show that overexpression of EGFR or EGFRvIII in human glioma cells causes increased basal TF expression and that stimulation of EGFR by its ligand, EGF, leads to a marked dose-dependent up-regulation of TF. In all cases, increased TF expression led to accelerated plasma coagulation in vitro. EGFR-mediated TF expression depended most strongly on activator protein-1 (AP-1) transcriptional activity and was associated with c-Jun NH(2)-terminal kinase (JNK) and JunD activation. Restoration of PTEN expression in PTEN-deficient GBM cells diminished EGFR-induced TF expression by inhibiting JunD/AP-1 transcriptional activity. PTEN mediated this effect by antagonizing phosphatidylinositol 3-kinase activity, which in turn attenuated both Akt and JNK activities. These mechanisms are likely at work in vivo, as EGFR expression was highly correlated with TF expression in human high-grade astrocytoma specimens.

Topics: Brain Neoplasms; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Promoter Regions, Genetic; Proto-Oncogene Proteins c-jun; PTEN Phosphohydrolase; Signal Transduction; Thromboplastin; Transcription Factor AP-1; Transcription, Genetic; Up-Regulation

The leucine-rich and immunoglobulin-like domains (LRIG) gene family contains LRIG1, 2 and 3. LRIG1 is a negative regulator of EGFR, but little is known about the function of LRIG2. To determine the role of LRIG2 in the progression of glioma, we performed RNA interference-mediated knockdown of LRIG2 in a human glioma cell line (GL15). Downregulation of LRIG2 expression resulted in: rapid EGF-mediated loss of EGFR; decreased proliferation; G(0)/G(1) arrest; increased spontaneous apoptosis; enhanced cell adhesion and increased invasion capability of GL15 cells in vitro. These findings indicate that LRIG2 possesses distinct functions compared with LRIG1 and validate the attractiveness of LRIG2 as a target in glioma therapy.

Topics: Apoptosis; Cell Adhesion; Cell Cycle; Cell Line, Tumor; Central Nervous System Neoplasms; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Humans; Membrane Glycoproteins; RNA Interference; RNA, Neoplasm; RNA, Small Interfering; Transfection

Glioblastomas--uniformly fatal brain tumors--often have both monosomy of chromosome 10 and gains of the epidermal growth factor receptor (EGFR) gene locus on chromosome 7, an association for which the mechanism is poorly understood.. To assess whether coselection of EGFR gains on 7p12 and monosomy 10 in glioblastomas promotes tumorigenic epidermal growth factor (EGF) signaling through loss of the annexin A7 (ANXA7) gene on 10q21.1-q21.2 and whether ANXA7 acts as a tumor suppressor gene by regulating EGFR in glioblastomas.. Multidimensional analysis of gene, coding sequence, promoter methylation, messenger RNA (mRNA) transcript, protein data for ANXA7 (and EGFR), and clinical patient data profiles of 543 high-grade gliomas from US medical centers and The Cancer Genome Atlas pilot project (made public 2006-2008; and unpublished, tumors collected 2001-2008). Functional analyses using LN229 and U87 glioblastoma cells.. Associations among ANXA7 gene dosage, coding sequence, promoter methylation, mRNA transcript, and protein expression. Effect of ANXA7 haploinsufficiency on EGFR signaling and patient survival. Joint effects of loss of ANXA7 and gain of EGFR expression on tumorigenesis.. Heterozygous ANXA7 gene deletion is associated with significant loss of ANXA7 mRNA transcript expression (P = 1 x 10(-15); linear regression) and a reduction (mean [SEM]) of 91.5% (2.3%) of ANXA7 protein expression compared with ANXA7 wild-type glioblastomas (P = .004; unpaired t test). ANXA7 loss of function stabilizes the EGFR protein (72%-744% increase in EGFR protein abundance) and augments EGFR transforming signaling in glioblastoma cells. ANXA7 haploinsufficiency doubles tumorigenic potential of glioblastoma cells, and combined ANXA7 knockdown and EGFR overexpression promotes tumorigenicity synergistically. The heterozygous loss of ANXA7 in approximately 75% of glioblastomas in the The Cancer Genome Atlas plus infrequency of ANXA7 mutation (approximately 6% of tumors) indicates its role as a haploinsufficiency gene. ANXA7 mRNA transcript expression, dichotomized at the median, associates with patient survival in 191 glioblastomas (log-rank P = .008; hazard ratio [HR], 0.667; 95% confidence interval [CI], 0.493-0.902; 46.9 vs 74.8 deaths/100 person-years for high vs low ANXA7 mRNA expression) and with a separate group of 180 high-grade gliomas (log-rank P = .00003; HR, 0.476; 95% CI, 0.333-0.680; 21.8 vs 50.0 deaths/100 person-years for high vs low ANXA7 mRNA expression). Deletion of the ANXA7 gene associates with poor patient survival in 189 glioblastomas (log-rank P = .042; HR, 0.686; 95% CI, 0.476-0.989; 54.0 vs 80.1 deaths/100 person-years for wild-type ANXA7 vs ANXA7 deletion).. Haploinsufficiency of the tumor suppressor ANXA7 due to monosomy of chromosome 10 provides a clinically relevant mechanism to augment EGFR signaling in glioblastomas beyond that resulting from amplification of the EGFR gene.

Topics: Annexin A7; Brain Neoplasms; Cell Line, Tumor; Cell Transformation, Neoplastic; Chromosomes, Human, Pair 10; Chromosomes, Human, Pair 7; Epidermal Growth Factor; Epigenesis, Genetic; ErbB Receptors; Female; Gene Deletion; Gene Dosage; Gene Expression; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Genes, Tumor Suppressor; Glioblastoma; Humans; Loss of Heterozygosity; Male; Middle Aged; Monosomy; Mutation; PTEN Phosphohydrolase; RNA, Messenger; Signal Transduction; Survival Analysis

: Epidermal growth factor (EGF) signaling plays a pivotal role in gliomagenesis. The authors previously demonstrated that adenosine diphospate-ribosylation factor 6 (ARF6), a member of the Ras-related small guanosine-5'-triphospate-binding protein family, is required for EFA6A-induced glioma cell migration and invasion. However, the role of ARF6 in EGF signaling is unknown.. : The authors analyzed messenger RNA (mRNA) levels of ARF6 and EGF receptor (EGFR) in 16 high-grade glioma samples and in 6 low-grade glioma samples by reverse transcriptase-polymerase chain reaction analysis. To determine whether EGF induces ARF6 expression in human glioblastoma U87 cells through transcriptional regulation and EGFR activation, the levels of ARF6 were assayed in EGF-treated U87 cells that were preincubated with a transcriptional inhibitor (actinomycin D) and an EGFR tyrosine kinase inhibitor (PD153035), respectively. The downstream signaling of EGFR-mediated ARF6 up-regulation also was investigated using specific inhibitors of mitogen-activated protein kinase (MEK), phosphatidylinositol 3' kinase (PI3K), and Janus kinase 2. The involvement of SP1 in the downstream signaling was studied by using an SP1 inhibitor (mithramycin A). Small-interfering RNAs (siRNAs) targeting ARF6 were used to investigate the effects of ARF6 on EGF-mediated glioma cell proliferation.. : The results demonstrated that ARF6 and EGFR mRNA levels were elevated in glioma tissues. Furthermore, EGF stimulated ARF6 expression in U87 cells in a dose-dependent and time-dependant manner. This stimulation was caused by increased transcription of ARF6 and by activation of the MEK/extracellular signal-regulated kinase 1 and 2 (ERK1/2) and PI3K signaling pathways. It is noteworthy that SP1 was essential for EGF-induced ARF6 up-regulation. Finally, EGF-induced glioblastoma cell proliferation depended on ARF6, because the suppression of ARF6 by siRNA or by a dominant-negative mutant significantly inhibited EGF-induced cell proliferation.. : The results of the current study suggested that EGF-induced ARF6 expression plays a significant role in glioma cell proliferation. Cancer 2009. (c) 2009 American Cancer Society.

Topics: ADP-Ribosylation Factor 6; ADP-Ribosylation Factors; Cell Proliferation; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; RNA, Messenger; Signal Transduction; Sp1 Transcription Factor

Glioblastoma multiforme (GBM) is the most common and most malignant adult brain tumor. A characteristic of GBM is their highly invasive nature, making complete surgical resection impossible. The most common gain-of-function alteration in GBM is amplification, overexpression, and mutations of the epidermal growth factor receptor (EGFR). The constitutively activated mutant EGFR variant III (EGFRvIII), found in approximately 20% of GBM, confers proliferative and invasive advantage. The signaling cascades downstream of aberrant EGFR activation contributing to the invasive phenotype are not completely understood. Here, we show myristoylated alanine-rich protein kinase C substrate (MARCKS), previously implicated in cell adhesion and motility, contributes to EGFR-mediated invasion of human GBM cells. EGFRvIII-expressing or EGF-stimulated human GBM cells increased expression, phosphorylation, and cytosolic translocation of MARCKS in a protein kinase C-alpha-dependent manner. Down-regulation of MARCKS expression with small interfering RNA in GBM cells expressing EGFRvIII led to decreased cell adhesion, spreading, and invasion. Elucidation of mechanisms that promote EGFRvIII-mediated tumorigenesis in GBM, such as MARCKS, provides additional understanding and potential biological targets against this currently terminal human cancer.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Myristoylated Alanine-Rich C Kinase Substrate; Neoplasm Invasiveness; Protein Kinase C-alpha; RNA, Small Interfering; Signal Transduction; Transfection

Increased transcriptional activity of beta-catenin resulting from Wnt/Wingless-dependent or -independent signaling has been detected in many types of human cancer, but the underlying mechanism of Wnt-independent regulation remains unclear. We demonstrate here that EGFR activation results in disruption of the complex of beta-catenin and alpha-catenin, thereby abrogating the inhibitory effect of alpha-catenin on beta-catenin transactivation via CK2alpha-dependent phosphorylation of alpha-catenin at S641. ERK2, which is activated by EGFR signaling, directly binds to CK2alpha via the ERK2 docking groove and phosphorylates CK2alpha primarily at T360/S362, subsequently enhancing CK2alpha activity toward alpha-catenin phosphorylation. In addition, levels of alpha-catenin S641 phosphorylation correlate with levels of ERK1/2 activity in human glioblastoma specimens and with grades of glioma malignancy. This EGFR-ERK-CK2-mediated phosphorylation of alpha-catenin promotes beta-catenin transactivation and tumor cell invasion. These findings highlight the importance of the crosstalk between EGFR and Wnt pathways in tumor development.

Topics: alpha Catenin; Amino Acid Sequence; beta Catenin; Binding Sites; Casein Kinase II; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Glioblastoma; Humans; Molecular Sequence Data; Neoplasm Invasiveness; Phosphorylation; Phosphoserine; Protein Binding; Transcriptional Activation

Epidermal growth factor (EGF) receptor-mediated cell migration plays a vital role in invasion of many tumor types. EGF receptor ligands increase invasiveness in vivo, but it remains unclear how consequent effects on intrinsic cell motility behavior versus effects on extrinsic matrix properties integrate to result in net increase of translational speed and/or directional persistence of migration in a 3D environment. Understanding this convolution is important for therapeutic targeting of tumor invasion, as key regulatory pathways for intrinsic versus extrinsic effects may not be coincident. Accordingly, we have undertaken a quantitative single-cell imaging study of glioblastoma cell movement in 3D matrices and on 2D substrata across a range of collagen densities with systematic variation of protease-mediated matrix degradation. In 3D, EGF induced a mild increase in cell speed and a strong increase in directional persistence, the latter depending heavily on matrix density and EGF-stimulated protease activity. In contrast, in 2D, EGF induced a similarly mild increase in speed but conversely a decrease in directional persistence (both independent of protease activity). Thus, the EGF-enhanced 3D tumor cell migration results only partially from cell-intrinsic effects, with override of cell-intrinsic persistence decrease by protease-mediated cell-extrinsic reduction of matrix steric hindrance.

Topics: Brain Neoplasms; Cell Line, Tumor; Cell Membrane; Cell Movement; Collagen; Epidermal Growth Factor; Glioblastoma; Green Fluorescent Proteins; Humans; Ligands; Matrix Metalloproteinase 14; Models, Biological; Peptide Hydrolases; rac1 GTP-Binding Protein; Time Factors

To generate and evaluate a modular recombinant transporter (MRT) for targeting 211 At to cancer cells overexpressing the epidermal growth factor receptor (EGFR).. The MRT was produced with four functional modules: (1) human epidermal growth factor as the internalizable ligand, (2) the optimized nuclear localization sequence of simian vacuolating virus 40 (SV40) large T-antigen, (3) a translocation domain of diphtheria toxin as an endosomolytic module, and (4) the Escherichia coli hemoglobin-like protein (HMP) as a carrier module. MRT was labeled using N-succinimidyl 3-[211 At]astato-5-guanidinomethylbenzoate (SAGMB), its 125 I analogue SGMIB, or with 131 I using Iodogen. Binding, internalization, and clonogenic assays were performed with EGFR-expressing A431, D247 MG, and U87MG.wtEGFR human cancer cell lines.. The affinity of SGMIB-MRT binding to A431 cells, determined by Scatchard analysis, was 22 nM, comparable to that measured before labeling. The binding of SGMIB-MRT and its internalization by A431 cancer cells was 96% and 99% EGFR specific, respectively. Paired label assays demonstrated that compared with Iodogen-labeled MRT, SGMIB-MRT and SAGMB-MRT exhibited more than threefold greater peak levels and durations of intracellular retention of activity. SAGMB-MRT was 10-20 times more cytotoxic than [211 At]astatide for all three cell lines.. The results of this study have demonstrated the initial proof of principle for the MRT approach for designing targeted alpha-particle emitting radiotherapeutic agents. The high cytotoxicity of SAGMB-MRT for cancer cells overexpressing EGFR suggests that this 211 At-labeled conjugate has promise for the treatment of malignancies, such as glioma, which overexpress this receptor.

Topics: Alpha Particles; Antigens, Polyomavirus Transforming; Astatine; Benzoates; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Nucleus; Confidence Intervals; Dihydropteridine Reductase; Diphtheria Toxin; Drug Carriers; Endosomes; Epidermal Growth Factor; ErbB Receptors; Escherichia coli Proteins; Glioblastoma; Guanidine; Guanidines; Hemeproteins; Humans; NADH, NADPH Oxidoreductases; Radioimmunotherapy

To investigate the effects of leucine-rich repeats and immunoglobulin-like domains 3 (LRIG3) on the biological activity of glioblastoma cell line GL15.. Glioblastoma GL15 cells were cultured and transfected with LRIG3-EGFP plasmid. The location of LRIG3 in GL15 cells was observed with confocal microscopy. The proliferation and invasiveness of GL15 cells were detected with methyl thiazolyl tetrazolium (MTT) and Transwell methods respectively; the expression of epidermal growth factor receptor (EGFR) and LRIG3 mRNA and protein were detected with reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot respectively.. After transfection with the plasmid LRIG-EGFP, LRIG3 fusion protein was found in cytoplasm of GL15 cells and cell proliferative and invasiveness were reduced. The expression of EGFR and LRIG3 varied with the duration of EGF treatment (100 ng/ml): the expression of EGFR decreased while the expression of LRIG3 increased as time prolonged.. LRIG3 can inhibit the proliferation and invasiveness of glioblastoma cells and may be used as a target gene in gene therapy of glioblastoma.

Topics: Brain Neoplasms; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Humans; Membrane Proteins; Neoplasm Invasiveness; Plasmids; Recombinant Fusion Proteins; RNA, Messenger; Transfection; Tumor Cells, Cultured

Leucine-rich repeat C4 (LRRC4) has been shown to inhibit glioma cell proliferation, however, little is known about the mechanism(s) underlying the action of LRRC4. Here, we show that two glioblstoma U251 cell clones stably expressing LRRC4 were established. LRRC4 expression significantly inhibited the expression of some cytokines and their receptors determined by microarray and Western blot assays, and dramatically reduced cytokine-induced AP-1, NF-kB, and CyclinD1 activation in glioma cells. Furthermore, LRRC4 expression in glioma cells significantly downregulated spontaneous and cytokine-induced expression of K-RAS and phosphorylation of c-Raf, ERK, AKT, NF-kBp65, p70S6K, and PKC, suggesting that LRRC4 inhibited receptor tyrosine kinase (RTK) signaling pathways. Moreover, treatment with bFGF, IGF1, or IGF2 stimulated LRRC4(-/-), but not the LRRC4(+), glioma cell proliferation, indicating that LRRC4 mitigated cytokine-stimulated proliferation in glioma cells. In addition, treatment of LRRC4(-/-) glioma cells with EGF, IGF2, or PDGF promoted long distance mobilization, but induced little migration in LRRC4(+) glioma cells, suggesting that LRRC4 retarded cytokine-promoted glioma cell migration in vitro. Finally, human vessel endothelial cells (ECV304) treated with VEGF grew, aligned and formed hollow tube-like structures in vitro. In contrast, LRRC4(+) ECV304 treated with VEGF failed to form vessel-tube structures. Collectively, LRRC4 expression inhibited the expression of some growth factors, cytokines and their receptors, and the capacity of glioma cells responding to cytokine stimulation, leading to inhibition of glioma cell proliferation. Conceivably, induction of LRRC4 expression may provide new intervention for human glioma in the clinic.

Topics: Blotting, Western; Cell Culture Techniques; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; Clone Cells; Cytokines; Down-Regulation; Endothelial Cells; Endothelium, Vascular; Epidermal Growth Factor; Formazans; Glioblastoma; Humans; Insulin-Like Growth Factor II; Kidney; Luciferases; Neovascularization, Pathologic; Nerve Tissue Proteins; Oligonucleotide Array Sequence Analysis; Platelet-Derived Growth Factor; RNA, Neoplasm; Tetrazolium Salts; Transfection; Umbilical Veins; Vascular Endothelial Growth Factor A

Patients with gliomas expressing high levels of epidermal growth factor receptor (EGFR) and plasminogen activator inhibitor-1 (PAI-1) have a shorter overall survival prognosis. Moreover, EGF enhances PAI-1 expression in glioma cells. Although multiple known signaling cascades are activated by EGF in glioma cells, we show for the first time that EGF enhances expression of PAI-1 via sequential activation of c-Src, protein kinase C delta (PKCdelta), and sphingosine kinase 1 (SphK1), the enzyme that produces sphingosine-1-phosphate. EGF induced rapid phosphorylation of c-Src and PKCdelta and concomitant translocation of PKCdelta as well as SphK1 to the plasma membrane. Down-regulation of PKCdelta abolished EGF-induced SphK1 translocation and up-regulation of PAI-1 by EGF; whereas, down-regulation of PKCalpha had no effect on the EGF-induced PAI-1 activation but enhanced its basal expression. Similarly, inhibition of c-Src activity by PP2 blocked both EGF-induced translocation of SphK1 and PKCdelta to the plasma membrane and up-regulation of PAI-1 expression. Furthermore, SphK1 was indispensable for both EGF-induced c-Jun phosphorylation and PAI-1 expression. Collectively, our results provide a functional link between three critical downstream targets of EGF, c-Src, PKCdelta, and SphK1 that have all been implicated in regulating motility and invasion of glioma cells.

Topics: Cell Line, Tumor; Epidermal Growth Factor; Gene Expression Regulation; Glioblastoma; Humans; NF-kappa B; Phosphotransferases (Alcohol Group Acceptor); Plasminogen Activator Inhibitor 1; Protein Kinase C-alpha; Protein Kinase C-delta; Proto-Oncogene Proteins pp60(c-src); Signal Transduction; STAT Transcription Factors; Transcription Factor AP-1

Glioblastoma multiforme (GBM) is the highest grade of astrocytoma. GBM pathogenesis has been linked to receptor tyrosine kinases and kinases further down signal-transduction pathways - in particular, members of the protein kinase C (PKC) family. The expression and activity of various PKC isoforms are increased in malignant astrocytomas, but not in non-neoplastic astrocytes. This suggests that PKC activity contributes to tumor progression. The level of PKC-eta expressed correlates with the degree of phorbol-12-myristate-13-acetate (PMA)-induced proliferation of two glioblastoma cell lines, U-1242 MG and U-251 MG. Normally, U-1242 cells do not express PKC-eta, and PMA inhibits their proliferation. Conversely, PMA increases proliferation of U-1242 cells that are stably transfected with PKC-eta (U-1242-PKC-eta). PMA treatment also stimulates proliferation of U-251 cells, which express PKC-eta. Here, we determined that extracellular signal-regulated kinase (ERK) and Elk-1 are downstream targets of PKC-eta. Elk-1-mediated transcriptional activity correlates with the PKC-eta-mediated mitogenic response. Pretreatment of U-1242-PKC-eta cells with inhibitors of PKC or MAPK/ERK kinase (MEK) (bisindolyl maleimide (BIM) or U0126, respectively) blocked both PMA-induced Elk-1 transcriptional activity and PMA-stimulated proliferation. An overexpressed dominant-negative PKC-eta reduced the mitogenic response in U-251 cells, as did reduction of Elk-1 by small interfering RNA. Taken together, these results strongly suggest that PKC-eta-mediated glioblastoma proliferation involves MEK/mitogen-activated protein (MAP) kinase phosphorylation, activation of ERK and subsequently of Elk-1. Elk-1 target genes involved in GBM proliferative responses have yet to be identified.

Topics: Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; ets-Domain Protein Elk-1; Extracellular Signal-Regulated MAP Kinases; Genes, fos; Genes, jun; Glioblastoma; Humans; Isoenzymes; Models, Biological; Phosphorylation; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Transcription, Genetic; Transfection

The A/G61 polymorphism located in the 5'UTR of the EGF gene has been found to be both a risk factor and a prognostic factor in glioblastoma (GBM), but the functional consequences have not been investigated. Here we show, in vitro, that this polymorphism is functional, in that the G allele promoter is 40% more active than the A variant (P < 0.001). However, analysis of a large series of 209 GBM patients and 214 control subjects did not confirm that A/G61 polymorphism is a significant risk factor for GBM, despite a trend for higher GG frequency in these patients. Furthermore, A/G61 polymorphism was not a prognostic factor for survival in GBM patients, although it does appear to affect progression-free survival.

Topics: 5' Untranslated Regions; Adult; Aged; Aged, 80 and over; Animals; Brain Neoplasms; Case-Control Studies; CHO Cells; Cricetinae; DNA, Neoplasm; Epidermal Growth Factor; Genetic Predisposition to Disease; Genotype; Glioblastoma; Humans; Middle Aged; Polymorphism, Genetic; Prognosis; Risk Factors; Survival Rate; Transfection

The majority of glioblastoma multiforme (GBM) cells express the epidermal growth factor receptor (EGFR). The present study evaluates the combination of temozolomide (TMZ), EGFR inhibition, and radiotherapy (RT) in GBM cell lines.. Human GBM cell lines U87, LN229, LN18, NCH 82, and NCH 89 were treated with various combinations of TMZ, RT, and the monoclonal EGFR antibody cetuximab. Responsiveness of glioma cells to the combination treatment was measured by clonogenic survival.. Overall, double and triple combinations of RT, TMZ, and cetuximab lead to additive cytotoxic effects (independent toxicity). A notable exception was observed for U87 and LN 18 cell lines, where the combination of TMZ and cetuximab showed substantial antagonism. Interestingly, in these two cell lines, the combination of RT with cetuximab resulted in a substantial increase in cell killing over that expected for independent toxicity. The triple combination with RT, cetuximab, and TMZ was nearly able to overcome the antagonism for the TMZ/cetuximab combination in U87, however only marginally in LN18, GBM cell lines.. It appears that EGFR expression is not correlated with cytotoxic effects exerted by cetuximab. Combination treatment with TMZ, cetuximab and radiation resulted in independent toxicity in three out of five cell lines evaluated, the antagonistic effect of the TMZ/cetuximab combination in two cell lines could indicate that TMZ preferentially kills cetuximab-resistant cells, suggesting for some cross-talk between toxicity mechanisms. Expression of EGFR was no surrogate marker for responsiveness to cetuximab, alone or in combination with RT and TMZ.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cell Survival; Cetuximab; Chemotherapy, Adjuvant; Dacarbazine; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Epidermal Growth Factor; Glioblastoma; Humans; Radiation Dosage; Temozolomide

Primary glioblastomas (GBMs) commonly overexpress the oncogene epidermal growth factor receptor (EGFR), which leads to increased Ras activity. FTA, a novel Ras inhibitor, produced both time- and dose-dependent caspase-mediated apoptosis in GBM cell lines. EGFR-mediated increase in 3H-thymidine uptake was inhibited by FTA. FACS analysis was performed to determine the percent of apoptotic cells. The sub-Go population of GBM cells was increased from 4.5 to 13.8% (control) to over 45-53.6% in FTA-treated cells within 24 h. Furthermore, FTA also increased the activities of both caspase-3 and -9, and PARP cleavage. Treatment of GBMs with FTA before or after EGF addition to the cultures blocked phosphorylation of Akt and mitogen-activated protein kinases (MAPK). FTA also significantly reduced the amount of EGF-induced Ras-GTP as reflected by a decrease in the level of Ras bound to Raf-RBD-GST. This study demonstrates that inhibition of Ras methylation may provide a therapeutic target for the treatment of GBMs overexpressing EGFR.

Topics: Antineoplastic Agents; Apoptosis; Caspase 3; Caspase 9; Caspases; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Farnesol; Glioblastoma; Humans; Mitogen-Activated Protein Kinases; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-akt; ras Proteins; Salicylates; Signal Transduction; Time Factors

Glioblastoma multiforme (GBM) is the most lethal form of brain cancer. With the available treatments, survival does not exceed 12-14 mo from the time of diagnosis. We describe a novel strategy to selectively induce the death of glioblastoma cells and other cancer cells that over-express the EGF receptor. Using a non-viral delivery vector that homes to the EGF receptor, we target synthetic anti-proliferative dsRNA (polyinosine-cytosine [poly IC]), a strong activator of apoptosis, selectively to cancer cells.. Poly IC was delivered by means of a non-viral vector: 25kDa polyethylenimine-polyethyleneglycol-EGF (PEI25-PEG-EGF). EGFR-targeted poly IC induced rapid apoptosis in the target cells in vitro and in vivo. Expression of several cytokines and "bystander killing" of untransfected tumor cells was detected in vitro and in vivo. Intra-tumoral delivery of the EGFR-targeted poly IC induced the complete regression of pre-established intracranial tumors in nude mice, with no obvious adverse toxic effects on normal brain tissue. A year after treatment completion the treated mice remain cancer-free and healthy. Similarly, non-viral delivery of poly IC completely eliminated pre-established breast cancer and adenocarcinoma xenografts derived from EGFR over-expressing cancer cell lines, suggesting that the strategy is applicable to other EGFR-over-expressing tumors.. The strategy described has yielded an effective treatment of EGFR over-expressing GBM in an animal model. If this strategy is translated successfully to the clinical setting, it may actually offer help to GBM patients. Moreover the elimination of two additional EGFR over-expressing cancers in vivo suggests that in principle this strategy can be applied to treat other tumors that over-express EGFR.

Topics: Adenocarcinoma; Animals; Apoptosis; Bystander Effect; Cell Line, Tumor; eIF-2 Kinase; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Humans; Imines; Mammary Neoplasms, Experimental; Mice; Neoplasm Transplantation; Poly I-C; Polyethylene Glycols; Polyethylenes; RNA, Double-Stranded; Time Factors; Transfection; Transplantation, Heterologous

Topics: Animals; Apoptosis; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Mice; Poly I-C; RNA, Double-Stranded

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and Akt are important regulators of the phosphatidylinositol 3-kinase (PI3K) pathway and thus are important to the regulation of a wide spectrum of tumor-related biological processes. Akt regulates several critical cellular functions, including cell cycle progression; cell migration, invasion, and survival; and angiogenesis. Decreased expression of PTEN and overexpression of the Akt proto-oncogene, which is located downstream of PI3K, have been shown in a variety of cancers, including glioblastoma. Novel small-molecule inhibitors of receptors and signaling pathways, including inhibitors of the PI3K pathway, have shown antitumor activity, but inhibitors of Akt have not been examined. In this study, we tested our hypothesis that the pharmacologic inhibition of Akt has an antiproliferative effect on gliomas. We showed that two newly developed Akt inhibitors, KP-372-1 and KP-372-2 (herein called KP-1 and KP-2), effectively inhibited the PI3K/Akt signaling cascade. KP-1 and KP-2 blocked both the basal and epidermal growth factor-induced phosphorylation of Akt Ser473 at 125 and 250 nmol/L, which, in turn, reduced the activation of intracellular downstream targets of Akt, including GSK-3beta and p70s6k. Furthermore, the treatment of U87 and U251 glioma cells with 125 to 250 nmol/L KP-1 and KP2 for 48 hours inhibited cell growth by approximately 50%. This decrease in cell growth stemmed from the induction of apoptosis. Collectively, these results provide a strong rationale for the pharmacologic targeting of Akt for the treatment of gliomas.

Topics: Apoptosis Regulatory Proteins; Cell Line, Tumor; Cell Proliferation; Central Nervous System Neoplasms; Enzyme Activation; Epidermal Growth Factor; Glioblastoma; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Heterocyclic Compounds, 4 or More Rings; Humans; Phosphorylation; Proto-Oncogene Mas; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; Serine; Signal Transduction; Tetrazoles

Glioblastoma multiforme is a primary brain tumor associated with extensive invasion into surrounding brain tissue. Matrix metalloproteinases (MMPs) and urokinase plasminogen activation (uPA) system are shown to be involved in tumor invasion as they help in degradation of extracellular matrix (ECM) proteins and thus assist in the movement of cells. MMP-2 and 9 were shown to be upregulated in gliomas, suggesting their involvement in invasion. Genistein and biochanin A are isoflavones commonly known as phytoestrogens and have some anticancer properties. We hypothesize that these two isoflavones can induce a lowering of tumor invasion by decreasing the activity of matrix degrading enzymes. In this study we investigated the effects of genistein and biochanin A on invasive activity of U87MG cells using the Calbiochem in vitro invasion assay system. Our results suggest that genistein and biochanin A induced a decrease in invasive activity of U87MG cells in a dose-related manner. Genistein also induced a decrease in EGF-stimulated invasion thereby implicating an involvement of EGF-mediated signaling in invasion. Our results also show that treatment of U87MG cells with the two isoflavones induced decreases in the enzymatic activity of MMP-9 and the protein levels of MT1-MMP and uPAR.

Topics: Brain Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Inhibitors; Enzyme Precursors; Epidermal Growth Factor; Extracellular Matrix; Genistein; Glioblastoma; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinases; Matrix Metalloproteinases, Membrane-Associated; Neoplasm Invasiveness; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator

The concept of tumor stem cells (TSCs) provides a new paradigm for understanding tumor biology, although it remains unclear whether TSCs will prove to be a more robust model than traditional cancer cell lines. We demonstrate marked phenotypic and genotypic differences between primary human tumor-derived TSCs and their matched glioma cell lines. Unlike the matched, traditionally grown tumor cell lines, TSCs derived directly from primary glioblastomas harbor extensive similarities to normal neural stem cells and recapitulate the genotype, gene expression patterns, and in vivo biology of human glioblastomas. These findings suggest that TSCs may be a more reliable model than many commonly utilized cancer cell lines for understanding the biology of primary human tumors.

Topics: Animals; Cell Line, Tumor; Cluster Analysis; Epidermal Growth Factor; Fibroblast Growth Factor 2; Gene Expression Profiling; Genome, Human; Genotype; Glioblastoma; Humans; Loss of Heterozygosity; Mice; Mice, SCID; Models, Biological; Neoplastic Stem Cells; Phenotype; Serum; Transcription, Genetic; Tumor Cells, Cultured

The main molecular genetic changes identified in glioblastomas are overexpression/amplification of the epidermal growth factor receptor (EGFR) gene and mutation/ deletion of the tumor suppressor PTEN gene. These two genetic changes both play important roles in glial tumorigenesis and progression. In this study, we demonstrated that wild-type PTEN transfection inhibited the growth and transforming ability of U87MG cells by 69.3% and 73.5%, respectively. On the other hand, antisense-EGFR transfection inhibited the growth and transforming phenotype of these cells by 50.3% and 46.8%, respectively. However, cotransfection of U87MG cells with wild-type PTEN and antisense EGFR constructs could inhibit the cellular growth by 91.7%. The transforming phenotype of these cells was completely inhibited. In addition, these cotransfected cells showed a differentiated form and expressed much lower telomerase activity than cells transfected with wild-type PTEN or antisense-EGFR alone. In summary, these results suggest that cotransfection is a better approach to suppress glioma cell growth than wild-type PTEN transfer or antisense-EGFR transfection alone. This approach may prove useful as an adjunct therapy in the treatment of glioblastomas.

Topics: Blotting, Western; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; DNA, Antisense; DNA, Complementary; Epidermal Growth Factor; Glial Fibrillary Acidic Protein; Glioblastoma; Humans; Immunohistochemistry; PTEN Phosphohydrolase; Reverse Transcriptase Polymerase Chain Reaction; Telomerase; Transfection

Sphingosine 1-phosphate (S1P), a lysophospholipid, is involved in various cellular processes such as migration, proliferation, and survival. To date, the impact of S1P on human glioblastoma is not fully understood. Particularly, the concerted role played by matrix metalloproteinases (MMP) and S1P in aggressive tumor behavior and angiogenesis remains to be elucidated.. To gain new insights in the effect of S1P on angiogenesis and invasion of this type of malignant tumor, we used microarrays to investigate the gene expression in glioblastoma as a response to S1P administration in vitro. We compared the expression profiles for the same cell lines under the influence of epidermal growth factor (EGF), an important growth factor. We found a set of 72 genes that are significantly differentially expressed as a unique response to S1P. Based on the result of mining full-text articles from 20 scientific journals in the field of cancer research published over a period of five years, we inferred gene-gene interaction networks for these 72 differentially expressed genes. Among the generated networks, we identified a particularly interesting one. It describes a cascading event, triggered by S1P, leading to the transactivation of MMP-9 via neuregulin-1 (NRG-1), vascular endothelial growth factor (VEGF), and the urokinase-type plasminogen activator (uPA). This interaction network has the potential to shed new light on our understanding of the role played by MMP-9 in invasive glioblastomas.. Automated extraction of information from biological literature promises to play an increasingly important role in biological knowledge discovery. This is particularly true for high-throughput approaches, such as microarrays, and for combining and integrating data from different sources. Text mining may hold the key to unraveling previously unknown relationships between biological entities and could develop into an indispensable instrument in the process of formulating novel and potentially promising hypotheses.

Topics: Cell Line, Tumor; Data Interpretation, Statistical; Databases, Bibliographic; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Lysophospholipids; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neovascularization, Pathologic; Oligonucleotide Array Sequence Analysis; Protein Interaction Mapping; Sphingosine

The novel fusion protein, DAB389EGF, composed of the catalytic and translocation domains of diphtheria toxin (DAB389) fused with a His-Ala linker to human epidermal growth factor (EGF) was tested for antiglioma efficacy in an in vivo model of human glioma.. Female athymic nude mice (ages 4-6 weeks) were inoculated s.c. with 10 million U87MG human glioma cells in the right flank. When tumor volumes reached approximately 100 mm3 (approximately 6-8 days), i.t. injections of saline, DAB389IL2, or DAB389EGF 1, 3, 5 or 10 microg in 50 microL were given every other day for three to six doses. Animals were monitored twice daily and tumor measurements were made by calipers.. The maximal tolerated dose (MTD) of DAB389EGF was 3 microg every other day. Above the MTD, animals experienced loss of activity, reduced oral intake, and dehydration. Blood chemistries confirmed elevated blood urea nitrogen, creatinine, aspartate transaminase, and alanine transaminase. Histopathology revealed renal tubular necrosis. At the MTD, tumor regression was seen in all animals. Relapses occurred in 4 of 16 (25%) of animals after 1 month. These tumors contained EGF receptor, were sensitive in vitro to DAB389EGF, and responded to a second course of i.t. DAB389EGF.. DAB389EGF fusion protein shows in vivo antiglioma efficacy in a s.c. tumor model and warrants further preclinical testing in an i.c. tumor model for eventual treatment of patients with recurrent or refractory EGF receptor-positive glioblastoma multiforme.

Topics: Animals; Blood Urea Nitrogen; Cell Line, Tumor; Diphtheria Toxin; Epidermal Growth Factor; Female; Glioblastoma; Glioma; Humans; Inhibitory Concentration 50; Kidney; Liver; Maximum Tolerated Dose; Mice; Mice, Inbred BALB C; Mice, Nude; Necrosis; Neoplasm Transplantation; Nitrogen; Protein Structure, Tertiary; Recombinant Fusion Proteins; Time Factors; Treatment Outcome

Glioblastoma multiforme, the most aggressive form of primary brain tumor in adults, is nearly universally fatal, with 5-year survivals of <5% (P. Kleihues and W. K. Cavenee, eds., pp. 1-314, Lyon: IARC, 2000). Alterations in the epidermal growth factor receptor (EGFR) are common events in many glioblastoma. We hypothesized that a polymorphism in the 5'-untranslated region of the epidermal growth factor (EGF) gene, a natural ligand of the EGFR, may play a role in the genesis of these malignant gliomas. We find that patients with the GA or GG genotype have higher tumoral levels of EGF, irrespective of EGFR status, that they are more likely to recur after surgery, and that they have a statistically significant shorter overall progression-free survival than patients with the AA genotype. These findings suggest that a single nucleotide polymorphism in EGF may play a role in the formation of glioblastomas, is a useful and powerful prognostic marker for these patients, and may be a target for tumor therapy.

Topics: 5' Untranslated Regions; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Humans; Polymorphism, Genetic

Cannabinoids, the active components of marijuana and their endogenous counterparts were reported as useful analgetic agents to accompany primary cancer treatment by preventing nausea, vomiting, and pain and by stimulating appetite. Moreover, they have been shown to inhibit cell growth and to induce apoptosis in tumor cells. Here, we demonstrate that anandamide, Delta(9)-tetrahydrocannabinol (THC), HU-210, and Win55,212-2 promote mitogenic kinase signaling in cancer cells. Treatment of the glioblastoma cell line U373-MG and the lung carcinoma cell line NCI-H292 with nanomolar concentrations of THC led to accelerated cell proliferation that was completely dependent on metalloprotease and epidermal growth factor receptor (EGFR) activity. EGFR signal transactivation was identified as the mechanistic link between cannabinoid receptors and the activation of the mitogen-activated protein kinases extracellular signal-regulated kinase 1/2 as well as prosurvival protein kinase B (Akt/PKB) signaling. Depending on the cellular context, signal cross-communication was mediated by shedding of proAmphiregulin (proAR) and/or proHeparin-binding epidermal growth factor-like growth factor (proHB-EGF) by tumor necrosis factor alpha converting enzyme (TACE/ADAM17). Taken together, our data show that concentrations of THC comparable with those detected in the serum of patients after THC administration accelerate proliferation of cancer cells instead of apoptosis and thereby contribute to cancer progression in patients.

Topics: ADAM Proteins; ADAM17 Protein; Amphiregulin; Apoptosis; Benzoxazines; Cannabinoids; Cell Division; Dronabinol; EGF Family of Proteins; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Glycoproteins; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Lung Neoplasms; Metalloendopeptidases; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Morpholines; Naphthalenes; Phosphorylation; Proto-Oncogene Proteins; RNA Interference; Signal Transduction; Transcriptional Activation; Tumor Cells, Cultured

Patients with glioblastoma multiforme have a poor prognosis due to recurrences originating from spread cells. The use of radionuclide targeting might increase the chance of inactivating single tumor cells with minimal damage to surrounding healthy tissue. As a target, overexpressed epidermal growth factor receptors (EGFR) may be used. A natural ligand to EGFR, the epidermal growth factor (EGF) is an attractive targeting agent due to its low molecular weight (6 kDa) and high affinity for EGFR. 177Lu (T(1/2) = 6.7 days) is a radionuclide well suited for treatment of small tumor cell clusters, since it emits relatively low-energy beta particles. The goal of this study was to prepare and preclinically evaluate both in vitro and in vivo the [177Lu]Bz-DTPA-EGF conjugate. The conjugate was characterized in vitro for its cell-binding properties, and in vivo for its pharmacokinetics and ability to target EGFR. [177Lu]Bz-DTPA-EGF bound to cultured U343 glioblastoma cells with an affinity of 1.9 nM. Interaction with EGFR led to rapid internalization, and more than 70% of the cell-associated radioactivity was internalized after 30 minutes of incubation. The retention of radioactivity was good, with more than 65% of the 177Lu still cell-associated after 2 days. Biodistribution studies of i.v. injected [177Lu]Bz-DTPA-EGF in NMRI mice demonstrated a rapid blood clearance. Most of the radioactivity was found in the liver and kidneys. The liver uptake was receptor-mediated, since it could be significantly reduced by preinjection of unlabeled EGF. In conclusion, [177Lu]Bz-DTPA-EGF seems to be a promising candidate for locoregional treatment of glioblastoma due to its high binding affinity, low molecular weight, and ability to target EGFR in vivo.

Topics: Animals; Cell Line, Tumor; Chelating Agents; Drug Evaluation, Preclinical; Epidermal Growth Factor; Glioblastoma; Humans; Lutetium; Mice; Mice, Inbred Strains; Pentetic Acid; Protein Binding; Radioisotopes; Radiopharmaceuticals; Sensitivity and Specificity; Tissue Distribution

Topics: Antineoplastic Agents; Brain Neoplasms; Clinical Trials as Topic; Disease-Free Survival; Endpoint Determination; Epidermal Growth Factor; Gefitinib; Glioblastoma; Humans; Neoplasm Recurrence, Local; Protein-Tyrosine Kinases; Quinazolines; Reproducibility of Results

EGF-receptors (EGFR) are overexpressed in gliomas, as well as in tumors of breast, lung, and urinary bladder. For this reason, EGFR may be an attractive target for both visualization and therapy of malignant tumors using radioactive nuclides. Natural ligand of EGFR, epidermal growth factor (EGF) is a small 53-amino-acid protein. Low molecular weight of EGF may enable better intratumoral penetration in comparison to antibodies. [111In]DTPA-EGF was proposed for the targeting of glioblastoma and breast cancer, and its tumor-seeking properties were confirmed in animal studies. The aim of this study was to evaluate how the substitution of heptadentate DTPA for octadentate benzyl-DTPA (Bz-DTPA) effects the biodistribution of indium-labeled human EGF (hEGF) in normal NMRI mice. [111In]DTPA-hEGF and [111In]Bz-DTPA-hEGF, obtained by the coupling of ITC-benzyl-DTPA to hEGF, were injected into the tail vein. At 0.5, 1, 4, and 24 hours postinjection, the animals were sacrificed, and radioactivity in different organs was measured. The blood clearance of both conjugates was fast. The uptake of both conjugates in the liver, spleen, stomach, pancreas, intestines, and submaxillary gland was most likely receptor-mediated. The uptake in a majority of organs was similar. However, indium uptake in the case of [111In]DTPA-hEGF was significantly higher in the kidneys and bones. In conclusion, [111In]Bz-DTPA-hEGF seems to have more favourable in vivo distribution in comparison to [111In]DTPA-hEGF.

Topics: Animals; Epidermal Growth Factor; ErbB Receptors; Female; Glioblastoma; Indium Radioisotopes; Isothiocyanates; Mice; Pentetic Acid; Radiopharmaceuticals

ADAMs (a disintegrin and metalloproteinases) are multifunctional molecules involved in cell-cell fusion, cell adhesion, membrane protein shedding, and proteolysis. In the present study, we examined the mRNA expression of 13 different ADAM species with putative metalloproteinase activity in human astrocytic tumors, nonneoplastic brain tissues, and other intracranial tumors by reverse transcriptase-polymerase chain reaction, and found that prototype membrane-anchored ADAM12 (ADAM12m) is predominantly expressed in glioblastomas. Real-time quantitative polymerase chain reaction indicated that the expression level of ADAM12m is remarkably at least 5.7-fold higher in glioblastomas (n = 16) than in nonneoplastic brain tissues (n = 6), low grade (n = 7) and anaplastic astrocytic tumors (n = 9) (P < 0.05 for each group), and intracranial neurinomas (n = 5) (P < 0.01). In situ hybridization showed that glioblastoma cells are responsible for the gene expression. By immunohistochemistry, ADAM12m was predominantly immunolocalized on the cell membranes of glioblastoma cells. Immunoblotting analysis demonstrated that ADAM12m is expressed as an activated N-glycosylated form of approximately 90 kd in glioblastoma tissues. There was a direct correlation between the mRNA expression levels of ADAM12m and proliferative activity (MIB1-positive cell index) of gliomas (r = 0.791, P < 0.0001; n = 32). Protein bands consistent with the soluble form of heparin-binding epidermal growth factor, a substrate of ADAM12m, were observed by immunoblotting in glioblastoma samples with the ADAM12m expression, and inhibited by treatment with ADAM inhibitor of the glioblastomas. These data demonstrate for the first time that among the 13 different ADAM species, ADAM12m is highly expressed in human glioblastomas, and suggest the possibility that ADAM12m plays a role in the prominent proliferation of the glioblastomas through shedding of heparin-binding epidermal growth factor.

Topics: ADAM Proteins; ADAM12 Protein; Brain; Brain Neoplasms; Cell Line, Tumor; Cell Membrane; Cell Proliferation; DNA Primers; Epidermal Growth Factor; Glioblastoma; Glycosylation; Heparin; Heparin-binding EGF-like Growth Factor; Humans; Immunoblotting; Immunohistochemistry; Immunoprecipitation; In Situ Hybridization; Intercellular Signaling Peptides and Proteins; Membrane Proteins; Metalloendopeptidases; Protein Binding; Receptors, Cell Surface; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors

Topics: Brain Neoplasms; Colorectal Neoplasms; DNA, Neoplasm; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Genes, erbB-1; Glioblastoma; Point Mutation; Quinazolines

An in-frame deletion of 801 bp in exons 2-7 (type III mutation) of the epidermal growth factor receptor (EGFR) is detected at high incidence in primary glioblastoma tumors. A proteomic approach was used to generate differential protein expression maps of fetal human astrocytes (FHA), human glioblastoma cell lines U87MG and U87MG expressing type III EGFR deletion (U87MGdeltaEGFR) that confers high malignancy to tumor cells. Two-dimensional gel electrophoresis followed by in-gel digestion of separated spots and protein identification by LC-MS-MS and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) identified 23 proteins expressed at higher levels or exclusively in FHA and 29 proteins expressed at higher levels or exclusively in U87MG cells. Three proteins, ubiquitin, cystatin B, and tissue transglutaminase (TTG), were upregulated in U87MGdeltaEGFR relative to U87MG. Four proteins highly expressed by U87MG cells, Hsp27, major vault protein, TTG, and cystatin B, were analyzed by Western blot, ELISA, or RT-PCR in cell extracts and in tissue samples of glioblastoma multiforme (GBM; grade IV), low-grade astrocytomas (grades I and II), and nonmalignant brain lesions. All four proteins were highly expressed in GBM tissues compared to nonmalignant brain. These proteins may be used as diagnostic or functional (e.g., multiple drug resistance, invasiveness) markers for glioblastoma tumors.

Topics: Astrocytes; Biomarkers, Tumor; Brain Neoplasms; Cystatin B; Cystatins; Electrophoresis, Gel, Two-Dimensional; Epidermal Growth Factor; Fetus; Gene Expression Regulation, Neoplastic; Glioblastoma; Heat-Shock Proteins; HSP27 Heat-Shock Proteins; Humans; Molecular Chaperones; Mutation; Neoplasm Proteins; Phosphorylation; Proteins; Proteomics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transglutaminases; Tumor Cells, Cultured; Ubiquitin; Up-Regulation; Vault Ribonucleoprotein Particles

The cytotoxicity of a diphtheria toxin-human epidermal growth factor fusion protein (DAB(389)EGF) was tested against 14 human glioma cell lines. After cells were cultured for 48 h with various concentrations of DAB(389)EGF, the percentage reduction in thymidine incorporation was determined. For 13 of 14 cell lines, potent cytotoxicity was observed, with IC(50)s of 0.4-50 pM. The epidermal growth factor receptor (EGFR) density of these cell lines was determined by immunofluorescence microscopy, flow cytometry, and radioligand binding. These assays correlated well with each other and demonstrated EGFR levels of 15,000-230,000/cell for 13 of 14 cell lines. The cell line U138MG, which lacked EGFR, was the only cell line insensitive to DAB(389)EGF. Linear regression analysis showed a good correlation between EGFR density and DAB(389)EGF sensitivity (P < 0.001) and between results of flow cytometry and radiolabeled binding assays of EGFR density (P = 0.01). DAB(389)EGF may have potential for intracranial therapy of EGFR-positive glioblastomas.

Topics: Brain Neoplasms; Carcinoma, Squamous Cell; Diphtheria Toxin; Drug Screening Assays, Antitumor; Epidermal Growth Factor; ErbB Receptors; Flow Cytometry; Glioblastoma; Humans; Inhibitory Concentration 50; Kinetics; Microscopy, Fluorescence; Recombinant Fusion Proteins; Tumor Cells, Cultured

Glioma cell migration is determined by a complex interplay between soluble motogens and extracellular matrix components. Several growth factors are thought to be involved in glioma cell migration; however, little is known about their motogenic potency relative to one another.. Using modified Boyden chamber assays, we compared the chemotactic effects of scatter factor/hepatocyte growth factor (SF/HGF), transforming growth factor (TGF)-alpha, TGF-beta1, TGF-beta2, epidermal growth factor (EGF), fibroblast growth factor (FGF)-1, FGF-2, insulin-like growth factor (IGF)-1, IGF-2, platelet-derived growth factor (PDGF)-AA, PDGF-BB, vascular endothelial growth factor (VEGF), pleiotrophin (PTN), and midkine (MK) in concentrations ranging from 1 pmol/L to 50 nmol/L on three different human glioblastoma cell lines. Checkerboard analyses distinguished between chemotaxis and chemokinesis. We further investigated the motogenic effects on human cerebral microvascular endothelial cells and analyzed receptor expression profiles.. SF/HGF was the most potent chemotactic factor for all three glioblastoma cell lines, inducing up to 33-fold stimulation of migration. TGF-alpha showed the second strongest effect (up to 17-fold stimulation), and FGF-1 was also chemotactic for all three glioblastoma cell lines analyzed (maximal 4-fold effect). EGF, FGF-2, IGF-1, IGF-2, TGF-beta1, and TGF-beta2 were chemotactic for one or two of the cell lines (2- to 4-fold effects), whereas PDGF-AA, PDGF-BB, VEGF, PTN, and MK had no effect. In contrast, the most potent stimulators of cerebral microvascular endothelial cell migration were PDGF-AA (4-fold) and PDGF-BB (6-fold).. The expression levels of SF/HGF and TGF-alpha as well as their respective receptors, MET and EGFR, are known to correlate with glioma malignancy grade. The particularly strong motogenic effects of these two growth factors suggest that they could be promising targets for an antimigratory component of glioma therapy, at least in comparison with the 12 other factors that were analyzed.

Topics: Angiogenesis Inducing Agents; Brain Neoplasms; Carrier Proteins; Cell Movement; Cerebrovascular Circulation; Chemotactic Factors; Cytokines; Endothelium, Vascular; Epidermal Growth Factor; Fibroblast Growth Factors; Glioblastoma; Growth Substances; Hepatocyte Growth Factor; Humans; In Vitro Techniques; Midkine; Mitogens; Platelet-Derived Growth Factor; Somatomedins; Transforming Growth Factors

The overexpression of epidermal growth factor receptors, EGFR, in glioblastomas is well documented. Hence, the EGFR can be used as target structure for a specific targeting of glioblastomas. Both radiolabeled anti-EGFR antibodies and the natural ligand EGF are candidate agents for targeting. However, EGF, which has a rather low molecular weight (6 kDa), might have better tissue penetration properties through both normal tissue and tumors in comparison with anti-EGF antibodies and their fragments. The aim of this study was to prepare and evaluate in vitro an EGF-based antiglioma conjugate with residualizing label. Human recombinant EGF (hEGF) was coupled to isothiocyanate-benzyl-DTPA. The conjugate was purified from unreacted chelator using solid-phase extraction and labeled with (111)In. The labeling yield was 87% +/- 7%. The label was reasonably stable; the transchelation of (111)In to serum proteins was about 5% after incubation at 37 degrees C during 24 hours. The obtained [(111)In]benzyl-DTPA-hEGF conjugate was characterized in vitro using the EGFR expressing glioma cell line U343MGaCl2:6. The binding affinity, internalization, and retention of the conjugate were studied. The conjugate had receptor specific binding and the radioactivity was quickly internalized. The intracellular retention of radioactivity after interrupted incubation with conjugate was 71% +/- 1% and 59% +/- 1.5% at 24 and 45 hours, respectively. The dissociation constant was estimated to 2.0 nM. The results indicate that [(111)In]benzyl-DTPA-hEGF is a potential candidate for targeting glioblastoma cells, possibly using locoregional injection.

Topics: Binding, Competitive; Cell Line, Tumor; Drug Delivery Systems; Drug Stability; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Humans; Indium Radioisotopes; Isothiocyanates; Kinetics; Pentetic Acid; Protein Binding; Protein Transport; Recombinant Proteins; Thiocyanates; Time Factors

Hyperactive epidermal growth factor receptor (EGFR) signaling, which promotes unregulated cell growth and inhibits apoptosis, is believed to contribute to clinical radiation resistance of glioblastoma multiforme (GBM). Blockage of the EGFR signalling pathways may offer an attractive therapeutic target to increase the cytotoxic effects of radiotherapy. We report the effects of ZD1839 ('Iressa'), a selective EGFR tyrosine kinase inhibitor on the radiation sensitivity of the U251 GBM cell line, which expresses high levels of EGFR. In radiation survival experiments, 5 microM of ZD1839 had a significant radiosensitizing effect and increased cell death was observed at doses of 5Gy in the presence of ZD1839. Dose and schedule of drug administration in combination with radiation appeared to be a crucial element to obtain radiosensitization of the cells. These studies suggest novel therapeutic strategies in the treatment of GBM.

Topics: Antineoplastic Agents; Brain Neoplasms; Cell Division; Cerebellum; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Glioblastoma; Humans; Phosphorylation; Placenta; Quinazolines; Radiation-Sensitizing Agents; Radiation, Ionizing; Radiotherapy Dosage; Time Factors; Tumor Cells, Cultured; Tumor Stem Cell Assay

To determine the acute and long-term effects of a therapeutic dose of brain radiation in a primate model, the authors studied the clinical, laboratory, neuroimaging, molecular, and histological outcomes in rhesus monkeys that had received fractionated whole-brain radiation therapy (WBRT).. Twelve 3-year-old male primates (Macaca mulatta) underwent fractionated WBRT (350 cGy for 5 days/week for 2 weeks, total dose 3500 cGy). Animals were followed clinically and with laboratory studies and serial magnetic resonance (MR) imaging. They were killed when they developed medical problems or neurological symptoms, lesions appeared on MR imaging, or at study completion. Gross, histological, and molecular analyses were then performed. Nine (82%) of 11 animals that underwent long-term follow up (> 2.5 years) developed neurological symptoms and/or enhancing lesions on MR imaging, which were defined as glioblastoma multiforme (GBM), 2.9 to 8.3 years after radiation therapy. The GBMs were categorized as either unifocal (three) or multifocal (six), and were located in the supratentorial (six), infratentorial (two), or both (one) cranial regions. Histological examination revealed distant, noncontiguous tumor invasion within the white matter of all nine animals harboring GBMs. Novel interspecies comparative genomic hybridization (three animals) uniformly showed deletions in the GBMs that corresponded to chromosome 9 in humans.. The high rate of GBM formation (82%) following a therapeutic dose of WBRT in nonhuman primates indicates that radioinduction of these neoplasms as a late complication of this therapy may occur more frequently than is currently recognized in human patients. The development of these tumors while monitoring the monkeys' conditions with clinical and serial MR imaging studies, and access to the tumor and the entire brain for histological and molecular analyses offers an opportunity to gather unique insights into the nature and development of GBMs.

Topics: Animals; Brain Neoplasms; DNA, Neoplasm; Dose-Response Relationship, Radiation; Epidermal Growth Factor; Glioblastoma; Interleukin-5; Interleukin-6; Macaca mulatta; Male; Neoplasms, Radiation-Induced; Nucleic Acid Hybridization; Tumor Suppressor Protein p53

To study the growth effects of epidermal Growth Factor (EGF) and the calcium channel antagonist nicardipine (NC) on U251 MG, a human malignant glioma cell lime, which has high-affinity EGF receptors.. The growth effects of EGF and NC on U251 MG cultured in serum-free and serum-containing (10% fetal bovine serum, FBS) media respectively were observed by MTT colorimeritric analysis.. (1) EGF significantly enhanced the growth of U251 MG in a dose-dependent manner in serum-free conditions. The near-maximal effect was obtained at 10 mg.ml-1. The addition of serum obscured this effect. (2) NC decreased U251 MG cells proliferation, especially in serum-containing media, and completely blocked the growth-stimulated effect of EGF. The combinated effects of EGF (10 ng.ml-1) and NC equaled those of NC alone.. When serum is absent U251 MG cells showed a pronounced mitogenic response to EGF in a dosedependent manner, which approximated that achieved with 10% FBS. NC suppressed the growth of U251 MG cells and completely blocked the growth-stimulated effects of EGF, it can be used as an adjuvent therapy for human malignant gliomas.

Topics: Antineoplastic Agents; Brain Neoplasms; Cell Division; Culture Media, Serum-Free; Epidermal Growth Factor; Glioblastoma; Humans; Nicardipine; Tumor Cells, Cultured

Amplification and/or mutations of the epidermal growth factor (EGF) receptor have been frequently reported in human malignant gliomas, the most common primary tumor of the adult central nervous system. We have analyzed a panel of established human glioma cell lines for EGF receptor expression. The EGF receptor was expressed in all of the glioma cell lines tested, with highest levels found in the cell line U343MG-a. In addition, various amounts of a truncated form of the EGF receptor were detected. The platelet-derived growth factor (PDGF) alpha receptor, analyzed for comparison, was expressed at low levels in human glioma cells, with the exception of U-118MG and U-373MG cells. The truncated form of the EGF receptor has been discussed as a constitutively active variant of the receptor. Using antibodies directed against the active form of the EGF receptor, we show here that the truncated variant of the EGF receptor in U343MG-a cells is not in the active conformation. However, the full-length EGF receptor, highly expressed in U343MG-a cells, was very rapidly activated following EGF treatment. In line with this, phosphorylation and activation of the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK) in U343MG-a cells required administration of EGF. Moreover, using highly specific riboprobes we observed that EGF signaling increased the Egr-1 mRNA concentration in human glioma cells within 30 min. The increase in the Egr-1 mRNA concentration was followed by a transient synthesis of the Egr-1 protein. Likewise, Egr-1 mRNA and protein concentrations were increased in U-118MG and U-373MG cells treated with PDGF. The synthesis of Egr-1 in human glioma cells as a result of EGF or PDGF stimulation indicates that Egr-1 may be an important "late" part of the EGF and PDGF-initiated signaling cascades suggesting that Egr-1 functions as a "third messenger" in glioma cells connecting growth factor stimulation with changes in gene transcription.

Topics: Astrocytoma; Brain Neoplasms; DNA-Binding Proteins; Early Growth Response Protein 1; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Glioblastoma; Glioma; Humans; Immediate-Early Proteins; MAP Kinase Signaling System; Neoplasm Proteins; Peptide Fragments; Platelet-Derived Growth Factor; Protein Conformation; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured

SHP-2 is a ubiquitously expressed non-transmembrane tyrosine phosphatase with two SH2 domains. Multiple reverse-genetic studies have indicated that SHP-2 is a required component for organ and animal development. SHP-2 wild-type and homozygous mutant mouse fibroblast cells in which the N-terminal SH2 domain was target-deleted were used to examine the function of SHP-2 in regulating Phosphatidylinositol 3-Kinase (PI3K) activation by growth factors. In addition, SHP-2 and various mutants were introduced into human glioblastoma cells as well as SHP-2(-/-) mouse fibroblasts. We found that EGF stimulation and EGFR oncoprotein (DeltaEGFR) expression independently induced the co-immunoprecipitation of the p85 subunit of PI3K with SHP-2. Targeted deletion of the N-terminal SH2 domain of SHP-2 severely impaired PDGF- and IGF-induced Akt phosphorylation. Ectopic expression of SHP-2 in U87MG gliobastoma cells elevated EGF-induced Akt phosphorylation, and the effect was abolished by mutation of its N-terminal SH2 domain. Likewise, the reconstitution of SHP-2 expression in the SHP-2(-/-) cells enhanced Akt phosphorylation induced by EGF while rescuing that induced by PDGF and IGF. Further lipid kinase activity assays confirmed that SHP-2 modulation of Akt phosphorylation correlated with its regulation of PI3K activation. Based on these results, we conclude that SHP-2 is required for mediating PI3K/Akt activation, and the N-terminal SH2 domain is critically important for a "positive" role of SHP-2 in regulating PI3K pathway activation.

Topics: Animals; Cell Line; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Growth Substances; Humans; Intracellular Signaling Peptides and Proteins; Mice; Mutation; Phosphatidylinositol 3-Kinases; Phosphorylation; Platelet-Derived Growth Factor; Protein Serine-Threonine Kinases; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; SH2 Domain-Containing Protein Tyrosine Phosphatases; Signal Transduction; Somatomedins; src Homology Domains; Transfection; Tumor Cells, Cultured

The objective of this work was to observe and analyze the effects of epidermal growth factor (EGF) and the calcium channel antagonist nicardipine on the growth of U251MG, a human malignant glioma cell line, which have high-affinity EGF receptors.. The growth effects of EGF and nicardipine on U251MG cultured in serum-free and serum-supplemented (10% fetal bovine serum, FBS) medium respectively were observed by MTT colorimeritric analysis.. EGF significantly enhanced the growth of U251MG cells in a dose-dependent manner in serum-free medium. The maximal effect was seen at 20 ng/ml. The effects of EGF approximated those obtained in 10% FBS. Nicardipine decreased U251MG cell proliferation, especially in serum-supplemented medium, and completely blocked the growth-stimulated effects of EGF. The combined effects of EGF (10 ng/ml) and nicardipine equaled those of nicardipine alone.. When serum was absent, the U251MG cells showed a pronounced mitogenic response to EGF in a dose-dependent manner, which approximated that achieved with 10% FBS. The addition of serum obscured this effect. Nicardipine suppressed the growth of U251MG cells and completely blocked the growth-stimulated effects of EGF may suggest a possible role of this drug as adjuvant therapy for human malignant gliomas.

Topics: Calcium Channel Blockers; Cell Division; Dose-Response Relationship, Drug; Epidermal Growth Factor; Glioblastoma; Humans; Nicardipine; Tumor Cells, Cultured

Hepatocyte growth factor/scatter factor (HGF/SF) contributes to the malignant progression of human gliomas. We investigated the effect of HGF/SF on matrix metalloproteinase-2 (MMP-2), membrane type 1 matrix metalloproteinase (MT1-MMP) and tissue inhibitors of metalloproteinases (TIMPs), expressions of c-Met/HGF receptor-positive human glioblastoma cells. Treatment of U251 human glioblastoma cells with HGF/SF resulted in enhanced secretion of MMP-2 with an increased level of the active form. This was accompanied by enhanced expression (2.5-fold) of mRNA specific for MMP-2. The stimulatory effect of HGF/SF on MMP-2 expression did not occur in the presence of herbimycin A, a protein tyrosine kinase inhibitor. MT1 -MMP, a cell-surface activator of proMMP-2, was also up-regulated by HGF/SF in a dose-dependent manner. By contrast, the level of TIMP- 1 mRNAs was not altered significantly and that of TIMP-2 was reduced mildly by the HGF/SF treatment, suggesting that HGF/SF may eventually modulate a balance between MMP-2 and TIMPs in favor of the proteinase activity in the glioma cell microenvironment. HGF/SF also stimulated MMP-2 expression of other glioblastoma cell lines. Since glioblastomas frequently co-express HGF/SF and its receptor, our results suggest that HGF/SF might contribute to the invasiveness of glioblastoma cells through autocrine induction of MMP-2 expression and activation.

Topics: Benzoquinones; Brain Neoplasms; Disease Progression; Enzyme Induction; Enzyme Inhibitors; Epidermal Growth Factor; Gelatinases; Gene Expression Regulation, Neoplastic; Glioblastoma; Glioma; Hepatocyte Growth Factor; Humans; Lactams, Macrocyclic; Matrix Metalloproteinase 2; Matrix Metalloproteinases, Membrane-Associated; Metalloendopeptidases; Neoplasm Invasiveness; Neoplasm Proteins; Protein-Tyrosine Kinases; Quinones; Recombinant Proteins; Rifabutin; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Stimulation, Chemical; Tumor Cells, Cultured

We used a genetic approach to characterize features of mitogen-activated protein kinase (MAPK) activation occurring as a consequence of expression of distinct erbB receptor combinations in transformed human cells. Kinase-deficient erbB proteins reduced epidermal growth factor (EGF)-induced tyrosine phosphorylation of endogenous Shc proteins and also reduced immediate and sustained EGF-induced ERK MAPK activities in human glioblastoma cells, although basal ERK MAPK activities were unaffected. Basal and EGF-induced JNK and p38 MAPK kinase activities were equivalent in parental cancer cells and EGFR-inhibited subclones. When ectopically overexpressed in murine fibroblasts and human glioblastoma cells, a constitutively activated human EGF receptor oncoprotein (deltaEGFR) induced EGF-independent elevation of basal ERK MAPK activity. Basal JNK MAPK kinase activity was also specifically induced by deltaEGFR, which correlated with increased phosphorylation of a 54-kDa JNK2 protein observed in deltaEGFR-containing cells. The JNK activities in response to DNA damage were comparably increased in cells containing wildtype EGFR or deltaEGFR. Consistent with the notion that transforming erbB complexes induce sustained and unregulated MAPK activities, coexpression of p185(neu) and EGFR proteins to levels sufficient to transform murine fibroblasts also resulted in prolonged EGF-induced ERK in vitro kinase activation. Transforming erbB complexes, including EGFR homodimers, deltaEGFR homodimers, and p185(neu)/EGFR heterodimers, appear to induce sustained, unattenuated activation of MAPK activities that may contribute to increased transformation and resistance to apoptosis in primary human glioblastoma cells.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Animals; Brain Neoplasms; Cell Line, Transformed; Cell Transformation, Neoplastic; Dimerization; DNA Damage; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Genes, erbB; Glioblastoma; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Neoplasm Proteins; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Multimerization; Protein Processing, Post-Translational; Protein Structure, Tertiary; Proteins; Receptor, ErbB-2; Recombinant Fusion Proteins; Sequence Deletion; Shc Signaling Adaptor Proteins; Src Homology 2 Domain-Containing, Transforming Protein 1; Transfection; Tumor Cells, Cultured

To investigate the biological features and immunophenotypes of human glioblastoma cell line SHG-44 after long term passage.. Immunohistochemistry and in situ hybridization were used to study the proliferative activity, intermediate filament protein coexistence, expressions of oncoprotein, angiogenic factors and cell cycle regulation factors.. After 130 to 150 passages, SHG-44 cells were weakly positive for glial fibrillary acidic protein (GFAP), but strongly positive for vimentin. The labeling index of Ki-67 and PCNA were 83.5% +/- 10.2% and 70.0% +/- 18.7% respectively. Overexpression of p21 ras, c-erbB-2, epidermal growth factor (EGF) and EGF receptor were obtained. Basic fibroblast growth factor (bFGF), FGF receptor, vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS) were also up-regulated in this cell line. p16, p53, cdk4 and cyclin D1 could be detected in the cells and their indices were 43.1% +/- 11.2%, 20.7% +/- 6.6%, 33.1% +/- 11.4% and 29.2% +/- 4.7% respectively.. Expressive abnormalities of these growth factors, their receptors and the above oncoproteins as well as disorders of cell cycle regulation contribute to the rapid growth and high degree of malignancy of this cell line.

Topics: Angiogenesis Inducing Agents; Cell Cycle Proteins; Cell Division; Endothelial Growth Factors; Epidermal Growth Factor; ErbB Receptors; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Immunohistochemistry; In Situ Hybridization; Intercellular Signaling Peptides and Proteins; Ki-67 Antigen; Lymphokines; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Receptor, ErbB-2; RNA, Messenger; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

It has long been recognized that some patients with low-grade astrocytoma may survive for many years, whereas in others the disease follows a more malignant course resulting in a short survival time, usually due to malignant transformation into higher-grade tumors.. The aim of this study was to investigate angiogenesis in the initial biopsy specimen of tumor tissue as a biological marker to identify patients with low-grade astrocytoma who are at high risk of malignant tumor transformation or death.. Tumor tissue was studied in 74 consecutively treated adult patients in whom a diagnosis of diffuse supratentorial hemispheric histologically proven fibrillary low-grade astrocytoma was made and who underwent surgery between January 1972 and January 1994. Studies were conducted using monoclonal antibodies to the antigens of the proliferation-associated Ki-67 (MIB-1), factor VIII, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF). The overall 5-year survival rate for the entire patient population was 65%, with a median survival time of 7.5 years. The total mean follow-up period was 6.1 years. All tumors showed a low proliferative potential at the time of the initial operation, as demonstrated by an MIB-1 labeling index of less than 1.5%. Patients with more than seven microvessels in tumor tissue (29 cases) had a shorter survival time (mean 3.8 years) than those with seven or fewer microvessels (mean survival 11.2 years). This difference in survival times was significant by univariate (p = 0.001) and stepwise multivariate analyses (p < 0.001). Tumors with a larger number of microvessels also had a greater chance of undergoing malignant transformation (p = 0.001). Similarly, significant staining for VEGF was correlated with shorter survival times when using univariate (p = 0.003) and multivariate (p = 0.008) analyses and with a greater chance of malignant transformation (p = 0.002). Patients with tumors staining positive for VEGF (39 individuals) had a median survival time of 5.3 years, and those with tumors negative for VEGF (35 patients) had a median survival time of 11.2 years. No association was observed between bFGF, EGF, and survival or malignant transformation. The stepwise multivariate analysis included histological and clinical variables simultaneously.. The authors have shown that microvessel density and VEGF levels are independent prognostic markers of survival in fibrillary low-grade astrocytoma. This finding leads them to propose that fibrillary diffuse low-grade astrocytoma is not a single pathological entity but is composed of a spectrum of tumors with differing propensities to undergo malignant transformation that is at least partly based on their inherent angiogenic potential.

Topics: Adolescent; Adult; Aged; Analysis of Variance; Antibodies, Monoclonal; Astrocytoma; Biomarkers, Tumor; Capillaries; Cell Division; Cell Transformation, Neoplastic; Coloring Agents; Endothelial Growth Factors; Epidermal Growth Factor; Factor VIII; Female; Fibroblast Growth Factor 2; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Ki-67 Antigen; Lymphokines; Male; Microcirculation; Middle Aged; Multivariate Analysis; Neovascularization, Pathologic; Prognosis; Supratentorial Neoplasms; Survival Rate; Treatment Outcome; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Venules

The novel quinazoline derivative 4-(3'-bromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P154) exhibited significant cytotoxicity against U373 and U87 human glioblastoma cell lines, causing apoptotic cell death at micromolar concentrations. The in vitro antiglioblastoma activity of WHI-P154 was amplified > 200-fold and rendered selective by conjugation to recombinant human epidermal growth factor (EGF). The EGF-P154 conjugate was able to bind to and enter target glioblastoma cells within 10-30 min via receptor (R)-mediated endocytosis by inducing internalization of the EGF-R molecules. In vitro treatment with EGF-P154 resulted in killing of glioblastoma cells at nanomolar concentrations with an IC50 of 813 +/- 139 nM, whereas no cytotoxicity against EGF-R-negative leukemia cells was observed, even at concentrations as high as 100 microM. The in vivo administration of EGF-P154 resulted in delayed tumor progression and improved tumor-free survival in a severe combined immunodeficient mouse glioblastoma xenograft model. Whereas none of the control mice remained alive tumor-free beyond 33 days (median tumor-free survival, 19 days) and all control mice had tumors that rapidly progressed to reach an average size of > 500 mm3 by 58 days, 40% of mice treated for 10 consecutive days with 1 mg/kg/day EGF-P154 remained alive and free of detectable tumors for more than 58 days with a median tumor-free survival of 40 days. The tumors developing in the remaining 60% of the mice never reached a size > 50 mm3. Thus, targeting WHI-P154 to the EGF-R may be useful in the treatment of glioblastoma multiforme.

Topics: Animals; Antineoplastic Agents; Apoptosis; Brain Neoplasms; Dose-Response Relationship, Drug; Epidermal Growth Factor; Glioblastoma; Humans; Mice; Mice, SCID; Quinazolines; Transplantation, Heterologous; Tumor Cells, Cultured

Glioblastoma multiforme is a highly invasive primary brain tumor with a disappointingly high local recurrence rate and mortality despite intensive multimodality treatment programs. Therefore, new agents that are capable of inhibiting the infiltration of normal brain parenchyma by glioblastoma cells are urgently needed. Here, we show that the novel quinazoline derivatives 4-(4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P131) and 4-(3'-bromo-4'hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P154) are potent inhibitors of glioblastoma cell adhesion and migration. Specifically, both compounds inhibited at micromolar concentrations: (a) integrin-mediated glioblastoma cell adhesion to the extracellular matrix proteins laminin, type IV collagen, and fibronectin; (b) integrin-independent epidermal growth factor-induced adhesion of glioblastoma cells to poly-L-lysine-coated tissue culture plates; (c) fetal bovine serum-induced polymerization of actin and actin stress fiber formation as well epidermal growth factor-stimulated formation of focal adhesion plaques in serum-starved glioblastoma cells; and most importantly, (d) glioblastoma cell migration in in vitro assays of tumor cell invasiveness using tumor cell spheroids and/or Matrigel-coated Boyden chambers. Further preclinical development of WHI-P131 and WHI-P154 may provide the basis for the design of more effective adjuvant chemotherapy programs for glioblastoma multiforme.

Topics: Actins; Antineoplastic Agents; Cell Adhesion; Epidermal Growth Factor; Glioblastoma; Humans; Integrins; Neoplasm Invasiveness; Quinazolines; Tumor Cells, Cultured

We previously reported that schwannoma-derived growth factor (SDGF), a member of heparin-binding epidermal growth factor (EGF) family, participates in autocrine pathways and promotes rat glioma cell growth. To investigate the potential role of similar molecules in human gliomas, we examined 7 human glioma cell lines and 11 glioblastoma specimens for expression of the human homologue of SDGF, amphiregulin (AR), as well as heparin-binding EGF-like growth factor (HB-EGF). Northern blot analysis revealed that only one cell line and no tumor specimens expressed AR mRNA. In contrast, HB-EGF mRNA was expressed in all human glioma cell lines and its level of expression was two- to five-fold higher than that of control brain tissues in 8 of 11 glioblastoma cases. Immunohistochemistry demonstrated that membrane-anchored HB-EGF (proHB-EGF) and EGFR were co-expressed in 44% of 34 human malignant gliomas. Introduction of exogenous HB-EGF (10 ng/ml) increased human glioma cell proliferation, and anti-HB-EGF blocking antibodies reduced the growth of glioma cells by 30-40%, confirming the presence of an autocrine loop. When added to the medium, transforming growth factor-alpha, basic fibroblast growth factor, or HB-EGF rapidly induced HB-EGF mRNA expression. These results indicate that HB-EGF and proHB-EGF contribute to the growth of human malignant glioma cells, most likely through autocrine and juxtacrine mechanisms.

Topics: Amphiregulin; Cell Division; EGF Family of Proteins; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Glioma; Glycoproteins; Growth Substances; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Mitosis; Recombinant Proteins; RNA, Messenger; Signal Transduction; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

Deregulation of signaling by the epidermal growth factor receptor (EGFR) is common in human malignancy progression. One mutant EGFR (variously named DeltaEGFR, de2-7 EGFR, or EGFRvIII), which occurs frequently in human cancers, lacks a portion of the extracellular ligand-binding domain due to genomic deletions that eliminate exons 2 to 7 and confers a dramatic enhancement of brain tumor cell tumorigenicity in vivo. In order to dissect the molecular mechanisms of this activity, we analyzed location, autophosphorylation, and attenuation of the mutant receptors. The mutant receptors were expressed on the cell surface and constitutively autophosphorylated at a significantly decreased level compared with wild-type EGFR activated by ligand treatment. Unlike wild-type EGFR, the constitutively active DeltaEGFR were not down-regulated, suggesting that the altered conformation of the mutant did not result in exposure of receptor sequence motifs required for endocytosis and lysosomal sorting. Mutational analysis showed that the enhanced tumorigenicity was dependent on intrinsic tyrosine kinase activity and was mediated through the carboxyl terminus. In contrast with wild-type receptor, mutation of any major tyrosine autophosphorylation site abolished these activities suggesting that the biological functions of DeltaEGFR are due to low constitutive activation with mitogenic effects amplified by failure to attenuate signaling by receptor down-regulation.

Topics: Amino Acid Sequence; Animals; Base Sequence; Brain Neoplasms; Cell Line; DNA Primers; Down-Regulation; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Humans; Mice; Mice, Nude; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptide Fragments; Phosphorylation; Phosphotyrosine; Recombinant Proteins; Signal Transduction; Transfection; Transplantation, Heterologous

Antisense oligodeoxynucleotides offer potential as therapeutic agents to inhibit gene expression. Recent evidence indicates that oligodeoxynucleotides designed to target specific nucleic acid sequences can interact nonspecifically with proteins. This report describes the interactive capabilities of phosphorothioate oligodeoxynucleotides of defined sequence and length with two essential protein tyrosine receptors, flk-1 and epidermal growth factor receptor (EGFR), and their effects on receptor signaling in a transfected and tumor cell line, respectively. Phosphorothioate oligodeoxynucleotides bound to the cell surface, as demonstrated by fluorescence-activated cell-sorter analyses (FACS), and perturbed receptor activation in the presence and absence of cognate ligands, EGF (EGFR) and vascular endothelial growth factor (flk-1), in phosphorylation assays. Certain phosphorothioate oligodeoxynucleotides interacted relatively selectively with flk-1 and partially blocked the binding of specific anti-receptor monoclonal antibodies to target sites. They stimulated EGFR phosphorylation in the absence of EGF but antagonized ligand-mediated activation of EGFR and flk-1. In vivo studies showed that a nonspecific phosphorothioate oligodeoxynucleotide suppressed the growth of glioblastoma in a mouse model of tumorigenesis. These results emphasize the capacity of phosphorothioate oligodeoxynucleotides to interact with cells in a sequence-selective nonantisense manner, while associating with cellular membrane proteins in ways that can inhibit cellular metabolic activities.

Topics: 3T3 Cells; Animals; Antineoplastic Agents; Base Sequence; Cell Membrane; Endothelial Growth Factors; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Humans; KB Cells; Lymphokines; Mice; Mice, Nude; Oligonucleotides, Antisense; Phosphorylation; Receptor Protein-Tyrosine Kinases; Receptors, Growth Factor; Receptors, Vascular Endothelial Growth Factor; Thionucleotides; Transplantation, Heterologous; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Photodynamic therapy (PDT) could be a useful adjuvant in glioblastoma treatment. The fact that epidermal growth factor (EGF) and its receptor are involved in glioblastoma growth control led us to investigate the relationships between EGF and PDT with respect to three different glioma cell lines (C6, T98 G, U87 MG) responsive to growth stimulation by EGF. Flow cytometric analysis revealed that each cell line expressed EGF receptors. PDT was then applied to the cells using haematoporphyrin derivative (HPD) as photosensitizer and argon laser irradiation. When cells were incubated for 2 h with HPD (0.1-10 micrograms/ml) and then laser-irradiated (lambda = 514 nm; energy density 25 J/cm2), all three cell lines showed photosensitivity. The median lethal dose was respectively 3, 4.5 and 2.7 micrograms/ml for C6, T98 G and U87 MG. EGF (2-50 ng/ml) had no effect on HPD- and laser-induced toxicity when added to cells before PDT, whereas toxicity decreased for all three cell lines when EGF was added after PDT. HPD (1-2 micrograms/ml, incubation times 30-180 min) also induced an increase in EGF receptor expression for the C6 line.

Topics: Brain Neoplasms; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Hematoporphyrins; Humans; Lasers; Photochemotherapy; Photosensitizing Agents; Tumor Cells, Cultured

Epidermal growth factor (EGF) and platelet-derived growth (PDGF) are suggested to be involved in the proliferation of human gliomas. We examined the effects of these growth factors on two human malignant glioma cell lines. Treatment of the A172 glioblastoma and the Hs683 glioma cell line with EGF and PDGF resulted in the tyrosine autophosphorylation, and hence activation, of the respective growth factor receptors. In addition, both cell lines responded to EGF and PDGF with increased deoxyribonucleic acid (DNA) synthesis. Because the intrinsic protein tyrosine kinase activity of this class of growth factor receptors is indispensable for their functioning, we tested the effects of specific protein tyrosine kinase inhibitors on growth factor-induced DNA synthesis and glioma cell proliferation. Genistein inhibited both EGF- and PDGF-stimulated autophosphorylation of the receptors and induction of DNA synthesis. However, genistein seemed to be cytotoxic to the cells. The tyrphostins RG 50875 and RG 13022 dose-dependently inhibited DNA synthesis induced by EGF, PDGF, and serum. RG 13022 completely blocked the EGF- and PDGF-induced DNA synthesis at a concentration of 50 mumol/L. The tyrphostins showed no selectivity in blocking either EGF or PDGF signaling. With concentrations up to mumol/L, no cytotoxic side effects of the tyrphostins were observed. Both tyrphostins also inhibit serum-driven cell growth in a dose-dependent manner. These results support the hypothesis that activated protein tyrosine kinase receptors are involved in the proliferation of A172 and Hs683 glioma cells. Selective inhibitors of protein tyrosine kinases, therefore, might have the potential to contribute to the treatment of growth factor-dependent gliomas.

Topics: Antineoplastic Agents; Brain Neoplasms; Cell Division; Cell Survival; DNA Replication; Dose-Response Relationship, Drug; Epidermal Growth Factor; Genistein; Glioblastoma; Glioma; Humans; Isoflavones; Platelet-Derived Growth Factor; Protein-Tyrosine Kinases; Tumor Cells, Cultured

Monocytes/macrophages frequently infiltrate malignant gliomas and play a central role in the tumor-associated immune response as they process tumor antigen and present it to T-lymphocytes. Findings have accumulated that peripheral blood monocytes leaving the cerebral circulation become microglial cells and vice versa and that monocytes/macrophages may stimulate malignant tumor growth by some unknown mechanism. Most malignant gliomas express growth factor receptors, for example epidermal growth factor receptor (EGFR). The aim of this study was to determine whether peripheral blood monocytes of glioma patients release EGF, the appropriate ligand of gliomacell membrane-bound EGFR. Long-term cultured peripheral blood monocytes from 14 patients with malignant gliomas were compared to those from 12 controls (seven with nontumorous disease and five healthy individuals). Using an enzyme-linked immunosorbent assay for EGF, the EGF content of cell culture supernatants was determined at Days 7, 21, and 100 of culture. The EGF content (mean +/- standard error) of supernatants was 5.9 +/- 0.2 pg/ml/10(3) glioma monocytes versus 1.3 +/- 0.1 pg/ml/10(3) control monocytes at Day 7 of culture, 22.9 +/- 0.8 pg/ml/10(3) glioma monocytes versus 1.8 +/- 0.9 pg/ml/10(3) control monocytes at Day 21 of culture, and 23.4 +/- 0.7 pg/ml/10(3) glioma monocytes, and below detection levels for control monocytes at Day 100 of culture. Steroid treatment of glioma patients did not influence the EGF release of cultured monocytes. These data indicate that glioblastoma-associated peripheral blood monocytes may be distinct from those of healthy individuals. Moreover, this study indicates that subtypes of glioma-associated peripheral blood monocytes may support immunosuppression and promote growth of malignant glioma by releasing unusually high amounts of EGF.

Topics: Brain Neoplasms; Epidermal Growth Factor; Female; Glioblastoma; Humans; Immunohistochemistry; Male; Middle Aged; Monocytes

Ethanol inhibits the tyrosine autophosphorylation of the insulin-like growth factor (IGF)-I receptor, an action that correlates with the inhibition of IGF-I-stimulated cell proliferation [J. Biol. Chem. 268:21777-21782 (1993)]. In the current study, the IGF-I-dependent proliferation of mouse BALB/c3T3 cells was completely inhibited by ethanol, but the growth of BALB/c3T3 cells that overexpress the IGF-I receptor (p6 cells) was only partially inhibited by ethanol BALB/ c3T3 cells that simultaneously overexpress both the IGF-I receptor and IGF-I were insensitive to growth inhibition by ethanol. In p6 cells, increasing concentrations of IGF-I overcame the inhibition of IGF-I receptor tyrosine autophosphorylation in the presence of ethanol. The importance of the IGF-I receptor as a specific target for ethanol was further investigated in C6 rat giloblastoma cells that respond mitogenically to both epidermal growth factor (EGF) and IGF-I. The mitogenic response of C6 cells to EGF was abrogated in cells expressing antisense mRNA to the IGF-I receptor. Thus, EGF action in these cells is dependent on activation of an IGF-I/IGF-I receptor autocrine pathway. Indeed, EGF stimulated an increase in IGF-I receptor levels by more than 100%. Ethanol completely inhibited the proliferation of C6 cells in response to either EGF or IGF-I. However, ethanol did not directly interfere with EGF receptor function, because EGF-induced cell proliferation was unaffected by ethanol when added exclusively during a 1-hr exposure to EGF. Ethanol did not interfere with the EGF-induced increase in IGF-I receptor expression. The addition of both EGF and IGF-I overcame the inhibitory action of ethanol. In conclusion, the potency of ethanol as an inhibitor of IGF-I-mediated cell proliferation correlates with the level of IGF-I receptors. In contrast to its effect on the IGF-I-receptor, ethanol has no direct effect on EGF receptor activation.

Topics: Animals; Cell Division; Cell Line; Epidermal Growth Factor; Ethanol; Glioblastoma; Insulin-Like Growth Factor I; Mice; Mice, Inbred BALB C; Rats; Receptor, IGF Type 1

We sought to determine whether hydroxyurea could accelerate the loss of amplified epidermal growth factor receptor (EGFR) genes from glioblastoma multiforme (GBM). There is good reason to think that elimination of amplified EGFR genes from GBMs will negatively impact tumor growth. Hydroxyurea has previously been shown to induce the loss of amplified genes from extrachromosomal double minutes (dmin) but not from chromosomal homogeneously staining regions.. Pulsed-field gel electrophoresis and Southern blot hybridization were used to demonstrate EGFR genes amplified as dmin. Giemsa-stained metaphase spreads were prepared in an attempt to visualize dmin. A GBM cell line containing amplified EGFR genes was treated continuously in vitro with 0 to 150 mumol/L hydroxyurea, and slot blot analysis was used to show the loss of amplified EGFR genes.. Amplified EGFR genes were found on dmin in 4 of 11 (36%) fresh human GBM biopsy specimens. None of the GBMs contained EGFR genes amplified as homogeneously staining regions. Amplified dmin were not microscopically visible when stained with Giemsa because of their small size. Slot blot analysis showed that these low doses of hydroxyurea accelerated the loss of amplified EGFR genes in a dose- and time-dependent fashion. Pulsed-field gel electrophoresis and Southern blot analysis confirmed that EGFR gene loss was accompanied by amplified dmin loss in a dose-dependent fashion.. These studies suggest the potential use of low-dose hydroxyurea in the treatment of GBMs.

Topics: Aged; Animals; Blotting, Southern; Cell Division; Cells, Cultured; Chromosomes; Dose-Response Relationship, Drug; Electrophoresis; Epidermal Growth Factor; Female; Gene Amplification; Genes; Glioblastoma; Humans; Hydroxyurea; Mice; Mice, Inbred BALB C; Middle Aged

Stimulation of three human glioma cell lines with basic fibroblast growth factor (bFGF) led to the enhancement of cell growth and the rapid tyrosine phosphorylation of cellular proteins, including major substrates of 90 kD. A methyltransferase inhibitor, 5'-methylthioadenosine (MTA), inhibited dose dependently the bFGF-stimulated cell growth and protein tyrosine phosphorylation in glioma cells by blocking both receptor autophosphorylation and substrate phosphorylation, as shown by immunoblotting with antiphosphotyrosine antibodies and cross-linking bFGF to receptors. The antiproliferative activity of MTA correlated quantitatively with its potency as an inhibitor of bFGF-stimulated protein tyrosine kinase activity. The methyltransferase inhibitor MTA had no effect on either epidermal growth factor- or platelet-derived growth factor-stimulated protein tyrosine phosphorylation in glioma cells, but inhibited specifically bFGF-stimulated protein tyrosine kinase activity. The concentration of MTA required for inhibition of protein methylation correlated well with the concentration required for inhibition of bFGF-stimulated cell growth and protein tyrosine phosphorylation. Because MTA had no effect on numbers and dissociation constants of high- and low-affinity bFGF receptors, the inhibition of bFGF-stimulated bFGF receptor tyrosine kinase activity is not likely to be the result of a reduction in bFGF receptor and bFGF binding capacity. In fact, MTA delayed and reduced the internalization and nuclear translocation of bFGF, and the internalized bFGF was submitted to a limited proteolysis that converted it to lower molecular peptides whose presence remained for at least 22 hours. The effect of MTA on bFGF-stimulated tyrosine phosphorylation was immediate and readily reversible.

Topics: Antibodies; Cell Division; Cell Line; Cell Nucleus; Cross-Linking Reagents; Deoxyadenosines; Dose-Response Relationship, Drug; Epidermal Growth Factor; Fibroblast Growth Factor 2; Glioblastoma; Glioma; Humans; Immunoblotting; Iodine Radioisotopes; Methyltransferases; Neoplasm Proteins; Phosphorylation; Phosphotyrosine; Platelet-Derived Growth Factor; Precipitin Tests; Protein-Tyrosine Kinases; Receptors, Fibroblast Growth Factor; Thionucleosides; Tyrosine

Platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) induce the proliferation of glioma cells in vitro. Trapidil and suramin inhibit this growth factor-stimulated glioma cell growth, but the mechanisms are not fully understood. The effects of trapidil and suramin on PDGF- and EGF-induced early biochemical events in T98G cells were studied. PDGF induced a rapid increase of intracellular free calcium concentration ([Ca2+]i) in fura-2/acetoxymethyl ester-loaded single glioma (T98G) cells. This increase was completely inhibited by removal of extracellular Ca2+ with ethylene glycol bis(beta-aminoethyl ether)-N,N,N,N-tetraacetic acid but not by an L-type calcium channel blocker (nicardipine), suggesting that PDGF may cause calcium influx through voltage-independent calcium channels in T98G cells. Trapidil and suramin blocked the PDGF-induced calcium response and inhibited the PDGF-initiated tyrosine phosphorylation of the PDGF receptor as detected by Western blot analysis using an antibody specific for phosphotyrosine. Trapidil and suramin also inhibited EGF-initiated calcium response in T98G cells, but only partially inhibited EGF-initiated tyrosine phosphorylation at the same concentrations. Our results suggest that trapidil and suramin inhibit PDGF- and EGF-initiated early biochemical events, and thus suppress growth factor-induced cell proliferation.

Topics: Antineoplastic Agents; Calcium; Cell Division; Epidermal Growth Factor; Glioblastoma; Humans; Phosphorylation; Platelet Aggregation Inhibitors; Platelet-Derived Growth Factor; Suramin; Trapidil; Tumor Cells, Cultured; Tyrosine

The epidermal growth factor receptor (EGFR) gene is amplified in over 40% of primary human glioblastomas and overexpressed in the majority. The authors' investigations demonstrate that the function of the EGFR in glioblastomas is distinct from that in other human cancers because it does not appear to mediate the primary growth-promoting effect of EGF. Findings show that the level of EGFR expression does not directly predict the growth response to EGF, with growth stimulated in some cells but inhibited in others when cells were cultured in plastic dishes. On the other hand, when human glioblastoma cells were placed in soft agar cultures, the cell line expressing the highest levels of the EGFR demonstrated considerable colony formation in response to EGF treatment. In addition, cell lines with the highest EGFR levels were also more resistant to the growth-suppressive effects of retinoic acid when maintained in soft agar. These observations suggest that even though the overexpression of the EGFR did not confer a distinct growth advantage to glioma cells cultured on flat culture dishes, the ability of these cells to maintain anchorage-independent growth in soft agar especially in response to EGF and retinoic acid is facilitated. Because anchorage-independent growth is the best in vitro correlate to tumorigenicity, amplification and overexpression of the EGFR in human glioblastoma cells may be in part responsible for the tumorigenic potential of these cells.

Topics: Animals; Brain Neoplasms; Cell Division; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Poisson Distribution; Tretinoin; Tumor Cells, Cultured

Our earlier investigations of the biology of the epidermal growth factor receptor (EGFR) in human gliomas demonstrated that the level of EGFR expression did not directly predict the glioma growth response to EGF, suggesting that the function of the EGFR in glioblastomas might not be limited to mediating the growth effects of EGF. We conducted the current studies to investigate the function(s) of the EGFR not related to growth control in human gliomas. These investigations show that the EGFR mediates the stimulative effects of EGF on glial process extension and glial fibrillary acidic protein (GFAP) expression. In addition, the level of EGFR expression correlates inversely with glioma cell responsiveness to differentiation promoting agents (for example, nerve growth factor and transforming growth factor-beta) that act through transmembrane tyrosine kinase receptors. Thus, glioma lines with a high level of EGFR expression (for example, T-98G cells) responded to fewer differentiation promoting factors than lines with a low level of EGFR expression (such as U-373MG cells). Our results suggest that the EGFR in gliomas may participate in mediating the process extension and GFAP stimulative effects of both EGF and other differentiation promoting agents. These properties represent components of the differentiated state in glia because their expression is stimulated by dibutyryl cyclic adenosine monophosphate in normal astrocytes. The involvement of the EGFR in the expression of these glial specific properties suggests that the EGFR may play an important role in glial differentiation.

Topics: Animals; Astrocytes; Brain Neoplasms; Bucladesine; Cell Division; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; ErbB Receptors; Glial Fibrillary Acidic Protein; Glioblastoma; Growth Substances; Humans; Immunohistochemistry; Neuroglia; Rats; Rats, Sprague-Dawley; Tretinoin; Tumor Cells, Cultured

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is an endothelial cell-specific mitogen that is structurally related to platelet-derived growth factor (PDGF). Vascular endothelial growth factor/vascular permeability factor induces angiogenesis in vivo and may play a critical role in tumor angiogenesis. Using immunohistochemical analysis, the authors demonstrated the presence of VEGF/VPF protein in surgical specimens of glioblastoma multiforme and cultured glioma cells. By means of an enzyme-linked immunosorbent assay (ELISA) of cell supernatants, the authors showed that VEGF/VPF is variably secreted by all nine cultured human malignant glioma cell lines (CH-235MG, D-37MG, D-54MG, D-65MG, U-87MG, U-105MG, U-138MG, U-251MG, U-373MG) and by a single meningioma cell line (CH-157MN). An immunocytochemical survey of these cell lines revealed a cytoplasmic and cell-surface distribution of VEGF/VPF. In the U-105MG glioma cell line, VEGF/VPF secretion was induced with physiological concentrations of epidermal growth factor, PDGF-BB, or basic fibroblast growth factor, but not with PDGF-AA. Moreover, it was observed that activation of convergent growth factor signaling pathways led to increased glioma VEGF secretion. Similar results were obtained using these growth factor combinations in the D-54MG glioma cell line. The data obtained suggest a potential role for VEGF/VPF in tumor hypervascularity and peritumoral edema. These observations may lead to development of new therapeutic strategies.

Topics: Adult; Blotting, Western; Brain Neoplasms; Capillary Permeability; Electrophoresis, Polyacrylamide Gel; Endothelial Growth Factors; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Glioblastoma; Humans; Lymphokines; Meningioma; Middle Aged; Platelet-Derived Growth Factor; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The epidermal growth factor (EGF) receptor is a membrane bound tyrosine kinase whose activity is initiated by ligand binding. The malignant brain tumour glioblastoma frequently shows amplification and rearrangements of the EGF receptor gene that are associated with the synthesis of a constitutively activated tyrosine kinase, lacking amino acids 6-273 near the protein's N-terminus. When expressed in Chinese hamster ovary (CHO) cells, this mutant receptor (p140EGFR) displays ligand-independent tyrosine kinase activity, stimulates DNA synthesis, and promotes cell proliferation. Here, we investigate the subcellular location of p140EGFR in CHO cell transfectants as well as in human glioblastoma tumours. p140EGFR had an intracellular location that contrasted sharply with the plasma membrane location of the wild-type EGF receptor. Endoglycosidase H sensitivity analysis and the pattern of p140EGFR immunoreactivity suggested that the aberrant tyrosine kinase resided primarily in the endoplasmic reticulum. The half-life of p140EGFR in the endoplasmic reticulum was extended several-fold over that of the ligand-activated wild-type receptor. The altered subcellular location of p140EGFR in combination with its prolonged half-life suggest that this activated tyrosine kinase may escape the regulatory mechanisms utilized for the attenuation of wild-type receptor signaling. Therefore, the previously reported growth stimulatory property of the ligand-independent p140EGFR may be attributed to a sustained tyrosine kinase activity resulting from an altered subcellular location.

Topics: Animals; Cell Compartmentation; Cell Membrane; CHO Cells; Cricetinae; Cytoplasm; Epidermal Growth Factor; ErbB Receptors; Fluorescent Antibody Technique; Glioblastoma; Hexosaminidases; Mutation; Transfection

A paradigm has been established whereby mutant tyrosine kinase receptors such as the v-erbB and v-fms gene products function as oncoproteins in the absence of ligand. A spontaneously occurring deletional mutant of the human epidermal growth factor receptor (EGFR-vIII) has been isolated from astrocytic neoplasms and transforms NIH3T3 cells in the absence of ligand. The EGFRvIII is constitutively complexed with the majority of cellular GRB2, suggesting a link to the Ras-Mitogen-activated protein (MAP) kinase pathway (D. Moscatello, R. B. Montgomery, P. Sundareshan, H. McDanel, M. Y. Wong, and A. J. Wong, submitted for publication). In this report, we document that expression of EGFRvIII in fibroblasts is associated with downstream activation of mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (MEK) and modest activation of p42 and p44 MAP kinases. The presence of EGFRvIII suppresses activation of p42 and p44 MAP kinases by phorbol 12-myristate 13-acetate (PMA) and serum; however, MEK activation by PMA is not suppressed by EGFRvIII. Basal and PMA-stimulated MAP kinase activity in EGFRvIII-transfected cells is augmented by the tyrosine phosphatase inhibitor sodium vanadate. EGFR-vIII is capable of transducing downstream signals through MAP kinase as evidenced by activation of cytoplasmic phospholipase A2 at levels similar to that induced by intact EGFR. Our results suggest that EGFR-vIII constitutively activates downstream signal transduction through MAP kinase, and this chronic stimulation of the MAP kinase pathway may represent one means by which mutant EGFR transduces an oncogenic signal.

Topics: 3T3 Cells; Animals; Calcium-Calmodulin-Dependent Protein Kinases; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Humans; Kinetics; Mice; Mitogen-Activated Protein Kinase Kinases; Protein Kinases; Recombinant Proteins; Sequence Deletion; Tetradecanoylphorbol Acetate; Transfection; Tumor Cells, Cultured; Vanadates

In 75 gliomas and 31 meningiomas, mutations at the epidermal growth factor receptor (EGFR) gene locus were restricted to gliomas. The ligands of this receptor, epidermal growth factor and transforming growth factor alpha, lacked quantitative changes at their loci in gliomas and meningiomas. EGFR gene amplification occurred in astrocytomas, oligodendrogliomas, ependymomas and glioblastomas. The frequency of this mutation significantly increased with the malignancy grade and the patient's age. Especially in glioblastomas of individuals aged over 64 years, EGFR gene mutations were observed without chromosome-10-specific allele losses. This finding contradicts the hypothesis that deletion of one entire chromosome 10 regularly precedes EGFR gene amplification in primary glioblastomas of patients aged over 50 years. It was found that most individuals whose gliomas carry an EGFR gene mutation have a poor prognosis, comparable to that of glioblastoma patients even when the tumour is graded as benign.

Topics: Adolescent; Adult; Aged; Brain Neoplasms; Child; Child, Preschool; Chromosome Mapping; Epidermal Growth Factor; ErbB Receptors; Female; Gene Amplification; Glioblastoma; Glioma; Humans; Male; Meningeal Neoplasms; Meningioma; Middle Aged; Mutation; Prognosis; Remission Induction; Survival Rate; Transforming Growth Factor alpha

Loss of genetic material on chromosome 10 is regarded as a prominent feature in the genesis of glioblastomas. To use chromosome 10 deletions as diagnostic markers for glioblastomas we investigated, if the loss of chromosome 10 material could be restricted on the region 10q21-26. By PCR microsatellite analysis on frozen tissue and paraffin material from the ZULCH brain tumor collection we found (1) loss of heterozygosity in 10q21-26 in 75% of the investigated DNA from frozen tissue and (2) an interstitial loss in the region of the microsatellite marker D10S186. The combined immunohistochemical analysis of overexpression of EGFR, EGF and TGF alpha with LOH on chromosome 10 showed that chromosome 10 deletions are not exclusively bound to EGFR overexpression.

Topics: Brain Neoplasms; Chromosome Deletion; Chromosome Mapping; Chromosomes, Human, Pair 10; DNA, Neoplasm; DNA, Satellite; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Glioblastoma; Glioma; Humans; Immunohistochemistry; Paraffin; Polymerase Chain Reaction; Transforming Growth Factor alpha

The influence of five anti-hormone and/or anti-growth factor neutralizing antibodies on the in vitro proliferation of four human astrocytic tumor cell lines (U87, U138, U373, H4) is quantitatively described by means of a new tool which makes it possible to evaluate cell growth and cell clone architecture concomitantly. This tool relies upon the combined use of the digital cell image analyses of Feulgen-stained nuclei and the Delaunay and Voronoi mathematical triangulation and paving techniques. Of the five anti-hormone and/or anti-growth factors tested here, the anti-luteinizing hormone-releasing hormone (LHRH) antibody induced the most marked perturbation in the U138 and U373 cell lines, whereas this role was played by the anti-epidermal growth factor (EGF) antibody in the U87 and H4 cell lines. The anti-gastrin (G) antibody significantly modified the growth and/or cell clone architecture of the U138, U87 and H4 cell lines, as did the anti-transforming growth factor alpha (TGFalpha) antibody. The anti-transforming growth factor beta (TGFbeta) antibody modified the growth and/or cell clone architecture of the four cell lines under study. If the five antibodies are taken into consideration, the results strongly suggest that four (the anti-G, the anti-EGF, the anti-LHRH and the anti-TGFalpha) act as inhibitory agents on some glioma cell line proliferation, while the fifth one, i.e. the anti-TGFbeta, act as a stimulator of cell proliferation, perhaps by abrogating the inhibitory effects of TGFbeta on proliferation. A comparison of cell growth data with cell clone architecture characteristics provided further evidence of some specific influence exercised by a given hormone and/or growth factor on glioma cell proliferation. Indeed, the anti-LHRH antibody caused the most pronounced perturbations in the U138 and U373 cell clone architecture; this feature was observed in the H4 cell line and, to a lesser extent in the U87 one after the anti-EGF antibody had been used.

Topics: Antibodies, Monoclonal; Cell Division; Epidermal Growth Factor; Gastrins; Glioblastoma; Gonadotropin-Releasing Hormone; Humans; Neuroblastoma; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

The role of the low-affinity neurotrophin receptor (p75NTR) in signal transduction is undefined. Nerve growth factor can activate the sphingomyelin cycle, generating the putative-lipid second messenger ceramide. In T9 glioma cells, addition of a cell-permeable ceramide analog mimicked the effects of nerve growth factor on cell growth inhibition and process formation. This signaling pathway appears to be mediated by p75NTR in T9 cells and NIH 3T3 cells overexpressing p75NTR. Expression of an epidermal growth factor receptor-p75NTR chimera in T9 cells imparted to epidermal growth factor the ability to activate the sphingomyelin cycle. These data demonstrate that p75NTR is capable of signaling independently of the trk neurotrophin receptor (p140trk) and that ceramide may be a mediator in neurotrophin biology.

Topics: 3T3 Cells; Animals; Astrocytes; Ceramides; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Mice; Nerve Growth Factors; Proto-Oncogene Proteins; Rats; Receptor Protein-Tyrosine Kinases; Receptor, trkA; Receptors, Nerve Growth Factor; Recombinant Fusion Proteins; Signal Transduction; Sphingomyelins; Tumor Cells, Cultured

Hypervascularity, focal necrosis, persistent cerebral edema, and rapid cellular proliferation are key histopathologic features of glioblastoma multiforme (GBM), the most common and malignant of human brain tumors. By immunoperoxidase and immunofluorescence, we definitively have demonstrated the presence of vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFr) in five out of five human glioma cell lines (U-251MG, U-105MG, D-65MG, D-54MG, and CH-235MG) and in eight human GBM tumor surgical specimens. In vitro experiments with glioma cell lines revealed a consistent and reliable relation between EGFr activation and VEGF production; namely, EGF (1-20 ng/ml) stimulation of glioma cells resulted in a 25-125% increase in secretion of bioactive VEGF. Conditioned media (CM) prepared from EGF-stimulated glioma cell lines produced significant increases in cytosolic free intracellular concentrations of Ca2+ ([Ca2+]i) in human umbilical vein endothelial cells (HUVECs). Neither EGF alone or CM from glioma cultures prepared in the absence of EGF induced [Ca2+]i increases in HUVECs. Preincubation of glioma CM with A4.6.1, a monoclonal antibody to VEGF, completely abolished VEGF-mediated [Ca2+]i transients in HUVECs. Likewise, induction by glioma-derived CM of von Willebrand factor release from HUVECs was completely blocked by A4.6.1 pretreatment. These observations provide a key link in understanding the basic cellular pathophysiology of GBM tumor angiogenesis, increased vascular permeability, and cellular proliferation. Specifically, EGF activation of EGFr expressed on glioma cells leads to enhanced secretion of VEGF by glioma cells. VEGF released by glioma cells in situ most likely accounts for pathognomonic histopathologic and clinical features of GBM tumors in patients, including striking tumor angiogenesis, increased cerebral edema and hypercoagulability manifesting as focal tumor necrosis, deep vein thrombosis, or pulmonary embolism.

Topics: Brain Neoplasms; Endothelial Growth Factors; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Glioma; Humans; Immunohistochemistry; Lymphokines; Models, Biological; Neovascularization, Pathologic; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

To examine the mechanisms by which alpha-interferon (IFN-alpha) inhibits growth factor-mediated proliferative responses, we examined specific ligand-activated, receptor-dependent events. In direct ligand binding studies, we showed that IFN-alpha treatment of cells leads to a reduction in epidermal growth factor (EGF) receptor recognition at the cell surface, coupled with an alteration in the binding characteristics of EGF for its specific receptors. Specifically, the heterogeneity of binding exhibited by EGF was affected, and there was loss of the high affinity binding component. EGF-induced autophosphorylation of the EGF receptor was unaffected by IFN treatment. The trafficking of EGF-receptor complexes was followed using three-dimensional confocal microscopy. Confocal imaging revealed that the rapid internalization of EGF-receptor complexes was significantly reduced when cells were exposed to IFN. Accompanying the IFN-induced changes in receptor binding characteristics, we identified an alteration in EGF receptor gene expression; when cells were treated with IFN-alpha, elevated RNA levels specific for the EGF receptor were detected. Overall, IFN-alpha treatment inhibited EGF-induced cell proliferation. Our results imply that EGF-bound receptors that are unable to internalize are not fully competent with respect to signal regulation of both gene expression and growth. The data suggest that the signaling potential of the bound growth factor-receptor complex is apparently increased by an unspecified, species-specific, high affinity binding component. We propose that IFN treatment of responsive cell prevents the interaction of EGF-bound receptor with this component.

Topics: Blotting, Northern; Blotting, Western; Cell Division; Cell Line; Epidermal Growth Factor; ErbB Receptors; Fluorescein-5-isothiocyanate; Glioblastoma; Humans; Interferon Type I; Kinetics; Phosphoproteins; Phosphorylation; Phosphotyrosine; Recombinant Proteins; RNA, Neoplasm; Tumor Cells, Cultured; Tyrosine

Spheroids initiated directly from human primary gliomas were used to investigate the effects of EGF, bFGF, NGF and PDGF(bb) on cell proliferation, migration and invasion into foetal rat brain tissue. EGF increased tumour spheroid volume in 10 of 13 glioblastomas studied, whereas 5 of 11 tumours responded to bFGF. NGF increased the spheroid volume in 2 of 5 tumours. In 8 tumours, PDGF(bb) had no effect on tumour spheroid volume. An increase in BUdR-labelling indices confirmed that cell proliferation was responsible for the volume increase observed in stimulated spheroids. EGF stimulated cell migration in 5 and bFGF in 3 of 8 tumours studied. NGF stimulated cell migration in 1 of 5 glioblastomas, whereas 1 of 3 glioblastomas responded to PDGF(bb). The effects of growth factors on the invasion of spheroids prepared from the glioblastoma biopsy specimens were also studied in vitro using foetal rat brain aggregates as target tissue. EGF stimulated invasion in 7 of 8 glioblastomas studied, whereas bFGF stimulated invasion in 2 of these tumours. NGF or PDGF(bb) did not increase the invasiveness of the glioblastoma tissue. Our results represent the net effect of the growth factors on a complex tumour-cell population. We conclude that exogenously administered growth factors, EGF in particular, increase the cell proliferation as well as migratory and invasive capacities of cultured primary brain tumour biopsies in vitro.

Topics: Animals; Brain; Brain Neoplasms; Cell Division; Cell Movement; Epidermal Growth Factor; Fetus; Fibroblast Growth Factor 2; Glioblastoma; Glioma; Growth Substances; Humans; Neoplasm Invasiveness; Nerve Growth Factors; Platelet-Derived Growth Factor; Rats; Tumor Cells, Cultured

A strategy for improved treatment of malignant gliomas grade III-IV is presented. The strategy can briefly be described as surgical removal of the bulky tumor, high precision external irradiation of small brain volumes over and near the primary tumor area with high doses from proton beams, and thereafter treatment of spread cells with toxic radionuclides. Proton beams suitable for this are under development. The clinical effects of high single doses on malignant gliomas grade III-IV are presently tested with conventional gamma radiation. Targeting of spread glioma cells with toxic radionuclides tagged to epidermal growth factor, EGF, or to EGF-dextran is presently tested in experimental systems and can, in the near future, be tested in combination with local high doses of external proton radiation. The possibilities to combine proton beams with EGF-guided neutron capture therapy will be considered in a longer perspective.

Topics: Brain Neoplasms; Combined Modality Therapy; Epidermal Growth Factor; Glioblastoma; Humans; Iodine Radioisotopes; Protons; Radiotherapy, High-Energy

The gene amplification and expression of transforming growth factor-alpha (TGF-alpha) and the epidermal growth factor receptor (EGF-R) in human gliomas was determined by Southern blot analysis and a receptor binding study. Amplification of the EGF-R gene was demonstrated in 3 of 11 tumors examined. All were glioblastomas, whereas the TGF-alpha gene was amplified in 7 of 11 tumors, and 6 of the 7 were recurrent glioblastomas and anaplastic astrocytomas. A competitive binding study with iodinated EGF showed a TGF-alpha-like activity ranging from 1.6-31.5 ng of EGF/microgram protein. These results suggest a differential expression of EGF-R and TGF-alpha genes among untreated and recurrent malignant gliomas.

Topics: Binding, Competitive; Blotting, Southern; Brain Neoplasms; Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Glioblastoma; Glioma; Humans; Iodine Radioisotopes; Tumor Necrosis Factor-alpha

A variety of tumors with different histologic types are included in a group of brain tumors. Although each histologic type of tumor has its own range of malignancy, the prognosis seems to be affected by several clinical, histologic and cell-biological factors. For example, relative survival rate of patients with glioblastoma is lower if the patient is older than 50 or 60 years. The leptomeningeal dissemination of glioma cells is a sign of poor prognosis. The presence of necrotic foci in the astrocytic tumors suggests shorter astrocytic tumors suggests shorter survival. Using a monoclonal antibody to bromodeoxyuridine (BrdU), the growth activity of the tumor can be estimated by BrdU labeling index (BrdU-LI, %). Higher BrdU-LI is correlated with more malignant histologic features in astrocytic tumors. In meningiomas, higher BrdU-LI is correlated with a more frequent or rapid recurrence of the tumor. The significance of growth factor receptors and oncogene of growth factor receptors and oncogene products as a cell-biologic marker of malignancy was investigated with an immunohistochemical method. Transferrin receptor was demonstrated in all tumors, and epidermal growth factor in about 40% of astrocytic tumors. The immunoreaction to c-myc oncogene product was detected in most astrocytic tumors; with higher intensity in anaplastic astrocytomas and glioblastomas than in low-grade astrocytomas. The role of these markers in the prognosis of brain tumors is, however, still unclear. Total or subtotal resection of glioblastoma results in longer resection of glioblastoma results in longer survival. Both postoperative radiotherapy and chemotherapy are effective. However, maintenance of chemotherapy longer than longer than 2 years does not significantly improve the prognosis.

Topics: Age Factors; Antibodies, Monoclonal; Astrocytoma; Biomarkers, Tumor; Brain Neoplasms; Bromodeoxyuridine; Cell Cycle; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Humans; Meningioma; Oligodendroglioma; Oncogenes; Prognosis

Urinary protein obtained from a patient with a highly malignant brain tumor (astrocytoma, grade IV) was adsorbed to trimethylsilyl controlled-pore glass beads and selectively eluted with acetonitrile to yield a high molecular weight (HMW) human transforming growth factor (hTGF). This HMW hTGF promoted clonogenic cell growth in soft agar and competed for membrane receptors with mouse epidermal growth factor. After surgical resection of the tumor, no HMW hTGF was found in urine. HMW hTGF generated a human EGF (hEGF) radioimmunoassay competitive binding curve similar to that of hEGF and parallel to that of a highly purified HMW form of hEGF previously reported to be present in trace concentrations in normal human urine. Both hEGF and HMW hEGF were clonogenic in soft agar, and their clonogenic activity as well as that of HMW hTGF was inhibited by anti-hEGF serum. Both HMW hTGF and HMW hEGF had 20 to 25% of the radioreceptor binding activity of hEGF. HMW hTGF purified from the pooled urine of several patients with malignant astrocytomas and HMW hEGF purified from normal control urine comigrated at Mr 33,000. Thus, HMW hTGF was indistinguishable from HMW hEGF in terms of apparent molecular size, epidermal growth factor receptor binding activity, epidermal growth factor immunoreactivity, and clonogenic activity. Urinary HMW hEGF/hTGF may be of tumor cell origin or may represent a response of normal host tissues to the tumor or its products.

Topics: Brain Neoplasms; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; Glioblastoma; Humans; Male; Middle Aged; Molecular Weight; Peptides; Transforming Growth Factors