Ligand-binding Domain Of Epidermal Growth Factor Receptor Research Articles

Modulation of EGFR Activity by Molecularly Imprinted Polymer Nanoparticles Targeting Intracellular Epitopes.

In recent years, molecularly imprinted polymer nanoparticles (nanoMIPs) have proven to be an attractive alternative to antibodies in diagnostic and therapeutic applications. However, several key questions remain: how suitable are intracellular epitopes as targets for nanoMIP binding? And to what extent can protein function be modulated via targeting specific epitopes? To investigate this, three extracellular and three intracellular epitopes of epidermal growth factor receptor (EGFR) were used as templates for the synthesis of nanoMIPs which were then used to treat cancer cells with different expression levels of EGFR. It was observed that nanoMIPs imprinted with epitopes from the intracellular kinase domain and the extracellular ligand binding domain of EGFR caused cells to form large foci of EGFR sequestered away from the cell surface, caused a reduction in autophosphorylation, and demonstrated effects on cell viability. Collectively, this suggests that intracellular domain-targeting nanoMIPs can be a potential new tool for cancer therapy.

CSIG-08. EXTRACELLULAR DOMAIN MUTATIONS IN EGFR RESULT IN A HYPERSENSITIVE RECEPTOR

Abstract INTRODUCTION The Epidermal Growth Factor Receptor (EGFR) is a driver gene that is amplified in more than half of all glioblastomas. After the initial high copy-number amplification, secondary missense mutations in the extracellular, ligand binding domain of EGFR can evolve. We here performed analysis on these mutations to better understand their function. METHODS Activation of EGFR-mutation constructs was measured by western blot and high-throughput quantitative image analysis using EGFR and phospho-EGFR specific antibodies. RESULTS Amphiregulin (AREG) is a low affinity EGFR ligand and was able to only marginally activate EGFRwt at high concentrations. In contrast however, AREG strongly activated all extracellular domain (ECD) mutation constructs (R108K, A289V and G598V). As expected, EGF-stimulation resulted in a strong activation in both EGFRwt and ECD mutation constructs. Further dose response analysis showed that in EGFRwt, all high affinity EGFR ligands (EGF, TGFa, HB-EGF and BTC) resulted in a strong activation and all low affinity ligands AREG, EREG and EPGN resulted in a markedly weaker activation. All ECD mutations however, showed strong activation towards all EGFR ligands, including the low affinity ligands (40%-80% of EGF stimulation). The constitutively active, ligand independent EGFRvIII mutation did not respond to any of the ligands and was mainly found to have an intracellular localization. Interestingly, the presence of ECD mutations and EGFRvIII are inversely correlated: in three large datasets (TCGA, Belob and Intellance 2), the majority of samples expressing EGFRvIII do not express additional ECD mutations and vice versa. ECD mutations likely affect stability of the tethered, inactive conformation thereby increasing exposure of the dimerization domain (II), resulting in increased ligand-sensitivity. CONCLUSION These experiments show that EGFR containing extracellular missense mutations render the receptor more sensitive to stimulation by low affinity ligands. Glioblastomas evolve to either become independent of ligand (EGFRvIII) or become ligand-hypersensitive (ECD mutations).

EGF receptor plays a role in the mechanism of glutamine-mediated prevention of alcohol-induced gut barrier dysfunction and liver injury

Recent study indicated that glutamine prevents alcoholic tissue injury in mouse gut and liver. Here we investigated the potential role of Epidermal Growth Factor Receptor (EGFR) in glutamine-mediated prevention of ethanol-induced colonic barrier dysfunction, endotoxemia and liver damage. Wild-type and EGFR*Tg transgenic (expressing dominant negative EGFR) mice were fed 1–6% ethanol in Lieber-DeCarli diet. Gut permeability was measured by vascular-to-luminal flux of FITC-inulin, and junctional integrity assessed by confocal microscopy. Liver injury was evaluated by plasma transaminases, histopathology and triglyceride analyses. Glutamine effect on acetaldehyde-induced tight junction disruption was investigated in Caco-2 cell monolayers. Doxycycline-induced expression of EGFR* blocked glutamine-mediated prevention of ethanol-induced disruption of colonic epithelial tight junction, mucosal permeability and endotoxemia. Ethanol activated cofilin and disrupted actin cytoskeleton, which was blocked by glutamine in an EGFR-dependent mechanism. Ethanol down-regulated antioxidant gene expression and up-regulated cytokine and chemokine gene expression, which were blocked by glutamine in wild-type mice in the presence or absence of doxycycline, but not in EGFR*Tg mice in the presence of doxycycline. Histopathology, plasma transaminases, triglyceride and expression of chemokine and antioxidant genes indicated ethanol-induced liver damage, which were blocked by glutamine in an EGFR-dependent mechanism. Src kinase activity and extracellular ligand binding domain of EGFR are required for glutamine-mediated protection of barrier function in Caco-2 cell monolayers. Glutamine released metalloproteinases into the medium, and metalloproteinase inhibitors blocked glutamine-mediated protection of barrier function. Results demonstrate that EGFR plays an important role in glutamine-mediated prevention of alcoholic gut permeability, endotoxemia and liver damage.

Ligand-based virtual screening, molecular docking, QSAR and pharmacophore analysis of quercetin-associated potential novel analogs against epidermal growth factor receptor

The present study was to explore expectation and examination of therapeutic potential quercetin analogs as efficient anticancer agents against human epidermal growth factor receptor (EGFR), which is a consistent hallmark for moderating the non-small-cell lung carcinoma (NSCLC). Here, ligand-based virtual screening, pharmacophore approach and molecular docking were established as rational strategies for recognition of small analogs against the ligand binding domain of EGFR (PDB code: 1XKK). Adverse effects, toxicogenomics and pharmacokinetics reported that 10 candidates showed reliable consequences with less side effects and more efficient for target receptor. Protein–ligand interaction profiles revealed that the probable H-bonds, atomic-π contacts, salt bridges and van der Waals interactions sustain the complexity and stability of receptor structure; thus, they could complicate to generate single alteration acquired for drug resistance. In silico anticancer properties explain the lead scaffolds which are assumed to be flexible and experimentally proved chemicals. The overall consequences indicated that recognized leads could be utilized as reference skeletons for new inhibitors envisaging toward EGFR to ameliorate NSCLC and other malignant disorders.

Endothelial PAS domain-containing protein 1 confers TKI-resistance by mediating EGFR and MET pathways in non-small cell lung cancer cells

Mutations in epidermal growth factor receptor (EGFR) rendering it constitutively active is one of the major causes for metastatic non-small-cell lung cancer (NSCLC), and EGFR-targeted therapies utilizing tyrosine kinase inhibitors (TKIs) are often used clinically as the first-line treatment. But approximately half of NSCLC patients develop resistance to these therapies, where the MET proto-oncogene is amplified by EGFR through the hypoxia-inducible factor (HIF)-1α. Here we report that endothelial PAS domain-containing protein 1 (EPAS1), with 48% sequence identity to HIF-1α, specifically binds to TKI-resistant T790M EGFR, but not to wild-type EGFR, in NSCLC cell lines. Expression of EPAS1 enhances amplification of MET when simultaneously expressed with T790M EGFR but not with wild-type EGFR, and this enhancement is independent of ligand binding domain of EGFR. MET amplification requires EPAS1, since EPAS1 knock-down reduced MET levels. When NSCLC cells expressing T790M EGFR were treated with TKIs, reduced EPAS1 levels significantly enhanced the drug effect, whereas over-expression of EPAS1 increased the drug resistant effect. This EPAS1-dependent TKI-resistance was abolished by knocking-down MET, suggesting that EPAS1 does not cause TKI-resistance itself but functions to bridge EGFR and MET interactions. Our findings suggest that EPAS1 is a key factor in the EGFR-MET crosstalk in conferring TKI-resistance in NSCLC cases, and could be used as a potential therapeutic target in TKI-resistant NSCLC patients.

A patient-derived somatic mutation in the epidermal growth factor receptor ligand-binding domain confers increased sensitivity to cetuximab in head and neck cancer

BackgroundCetuximab is an epidermal growth factor receptor (EGFR)-blocking antibody that has been approved for the treatment of patients with head and neck squamous cell carcinoma (HNSCC) and metastatic colorectal cancer, but no predictive biomarkers of activity have been yet identified. Establishment of such biomarkers will help identify a subset of patients that will benefit from cetuximab therapy. MethodsIn this paper, we report on a patient with HNSCC who had a complete tumour regression following treatment with cetuximab given as a single agent after initial surgery and radiation therapy. The EGFR protein expression level, the EGFR gene copy number and the EGFR gene sequence were assessed from both normal and tumour tissues. ResultsBesides protein overexpression and gene amplification in the tumour tissue, sequencing of the EGFR gene from the patient revealed the presence of two somatic mutations, one in the kinase domain (R705G) and the other in the ligand binding domain (P546S). Cells that stably express these EGFR mutants were treated with cetuximab and their sensitivity to the drug was compared to cells expressing the wildtype gene. While P546S mutation sensitised NIH-3T3 cells to cetuximab, R705G had a marginal effect. The double mutant (P546S/R705G) behaved like the P546S mutant, indicating that the mutation in the kinase domain does not contribute to the increased sensitivity to cetuximab. No mutations were found in K-RAS or B-RAF genes and no HPV protein or DNA was detected in the tumour. This is the first report of a somatic mutation in the EGFR ligand binding domain that may contribute to increased sensitivity to cetuximab. ConclusionsOur results support a role for the P546S mutation in cetuximab sensitivity. Other factors including EGFR protein high copy number and protein overexpression may have also contributed to the observed response. The severity of a skin rash developed by this patient and its correlation with the antitumour activity does not exclude the involvement of the immune system (i.e. complement-mediated immune response) as well. The occurrence of the P546S mutation needs to be evaluated in HNSCC, as a well as a prospective evaluation of cetuximab anti-tumour activity in patients with tumours harbouring the mutation.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Disparity Between In Vivo Zirconium-89-Labeled Cetuximab Uptake and EGFR Expression

The epidermal growth factor receptor (EGFR) is overexpressed in a significant percentage of human malignancies and its expression is associated with tumor aggressiveness and treatment resistance. The monoclonal antibody cetuximab (IMC-C225) blocks the ligand binding domain of EGFR, preventing downstream signaling and is increasingly used in a clinical setting. To assess in vivo binding of cetuximab to EGFR, we developed a new imaging probe using cetuximab labeled with the PET isotope Zirconium-89.

Epitope mapping of epidermal growth factor receptor (EGFR) in lung cancer using immunohistochemistry: Fine tuning the protocols

21162 Background: Until now, immunohistochemistry was not able to become a reliable diagnostic approach for EGFR targeted therapies. The golden standard of the determination of EGFR protein expression in paraffin embedded cancer tissues is the EGFRpharmDXtm kit. Methods: Here we show data based on analysis of 110 lung adenocarcinomas, that the recommended protocol may not be optimal for ideal performance of the immunodetection, since microwave retrieval, extended primary antibody-incubation time and replacement of the developer reagent converted four EGFR-negative tumor into EGFR protein positive out of eight lung adenocarcinoma cases. Protocol modification improved the performance of another widely used EGFR-kit, Ventana's CONFIRM, where replacement of the protease antigen retrieval with microwave cooking converted several EGFR-negative tumors to strongly positive. Meanwhile both EGFR-kits detect EGFR expression (juxtamembrane domain) but do not provide information on the expression of epitopes critical from the point of view of targeted therapy. Results: We have developed two protocols, which can detect the ligand-binding (AB-10, BioMarkers) and C-terminal (AB- 335, Biogenex) cytoplasmic domains of the EGFR protein in paraffin embedded lung cancer tissues. We have shown, based on the analysis of more than 110 lung adenocarcinoma tissues, that the ligand binding domain of EGFR is rarely expressed while the C-terminal domain is ubiquitously expressed in EGFR-PharmDX and CONFIRM-positive cancers. The biological activity of EGFR can be characterized either by autophosphorylation of the receptor or by detection of divers phosphorylated downstream signaling components. We have found that unlike p1086 (detected by a Zymed antibody), the p1173 site of EGFR (identified by a rabbit monoclonal of Epitomics) can be detected 27/110 paraffin embedded lung adenocarcinomas. Conclusions: Using the tested antibody panel we can reliably determine the EGFR protein expression in paraffin embedded (lung)cancer tissues. This work was supported by Ministry of Education (NKFP1a-0024–05). No significant financial relationships to disclose.

Mutations Within the Kinase Domain and Truncations of the Epidermal Growth Factor Receptor Are Rare Events in Bladder Cancer: Implications for Therapy

It has previously been reported that the patient response to gefitinib depends on the presence of mutations within the kinase domain of epidermal growth factor receptor (EGFR) or the expression of its truncated form, EGFR variant III (EGFRvIII). The focus of this study was to determine if these alterations are present within the tyrosine kinase and ligand-binding domain of EGFR in urothelial carcinoma. The kinase domain found within exons 18 to 21 of the EGFR from 11 bladder cancer cell lines and 75 patient tumors were subjected to automated sequencing. EGFRvIII expression was determined by immunohistochemistry using a urothelial carcinoma tissue microarray, and its expression was subsequently verified by reverse transcription PCR, real-time PCR, and Western blot analysis, using an EGFRvIII-transfected glioblastoma cell line and glioblastoma tumors as positive controls. Our analysis failed to detect mutations within the tyrosine kinase domain of EGFR in the 11 cell lines and 75 patients tested. The initial analysis of EGFRvIII expression by immunohistochemistry revealed that at least 50% of the patient tumors expressed EGFRvIII in a urothelial carcinoma tissue microarray. Conflicting reports exist, however, regarding the extent of EGFRvIII expression in tissues owing to the specificity of the antibodies and the methodologies used. Therefore, we sought to validate this observation by reverse transcription PCR, real-time PCR, and Western blot analysis. In these assays, none of the samples were positive for EGFRvIII except for control transfectants and glioblastomas. When our results are taken together, we conclude that alterations within the tyrosine kinase domain and expression of EGFRvIII are rare events in bladder cancer. The present study has clinical implications in selecting tyrosine kinase inhibitors for the therapy of urothelial carcinoma.

The epidermal growth factor receptor (EGFR)-S442F mutant displays increased affinity for neuregulin-2β and agonist-independent coupling with downstream signalling events

The EGFR (epidermal growth factor receptor; ErbB1) is frequently the subject of genetic changes in human tumours which contribute to the malignant phenotype by altering EGFR signalling. Examples of such genetic changes include overexpression, extracellular domain deletions and point mutations, and small deletions in the tyrosine kinase domain. We hypothesized that a point mutation in one of the EGFR ligand-binding domains would increase the affinity of EGFR for NRG2beta (neuregulin-2beta), which is not a potent stimulus of signalling by EGFR-Wt (wild-type EGFR). This mutation would permit NRG2beta stimulation of EGFR signalling in settings in which NRG2beta does not normally do so. To test this hypothesis, we have generated and evaluated various EGFR alleles containing mutations at Val441 and Ser442. NRG2beta is a much more potent stimulus of the EGFR-S442F mutant than of EGFR-Wt. Furthermore, the affinity of NRG2beta for the EGFR-S442F mutant is greater than the affinity of NRG2beta for EGFR-Wt. Finally, the EGFR-S442F mutant constitutively suppresses apoptosis via phosphoinositide 3-kinase and Akt signalling but is not highly tyrosine phosphorylated in the absence of ligand. These results suggest that mutations in the EGFR ligand-binding domain in tumours may permit potent stimulation of EGFR signalling by ligands that are not normally potent EGFR agonists, thereby providing for a novel mechanism by which EGFR signalling may be deregulated. These results also suggest that novel EGFR mutations and signalling activities may be responsible for deregulated EGFR signalling in tumour cells.

Hydrogen peroxide inhibits colonic epithelial ion transport by MAP kinase and PI3‐kinase (PI3K) independently of activated epidermal growth factor receptor (EGFr)

Reactive oxygen species contribute to epithelial damage and dysfunction in inflammatory bowel disease (IBD). We have shown that hydrogen peroxide (H2O2) inhibits intestinal Ca2+-dependent ion transport by PI3K-and MAP kinase-dependent mechanisms, while also activating the EGFr. Here, we studied whether EGFr regulates H2O2-mediated inhibition of ion transport in colonic epithelial cells. Protein phosphorylation was measured in T84 colonic epithelial monolayers by Western blot, and ion transport was measured in Ussing chambers. H2O2 (500 μM) increased EGFr tyrosine phosphorylation and its association with the catalytic p110β subunit of PI3-K in T84 cells. However, neither an EGFr kinase inhibitor, tyrphostin AG1478 (1 μM), nor a neutralizing anti-EGFr antibody that blocks the EGFr ligand binding domain (5 μg/ml), had any significant effect on H2O2-induced activation of ERK or the PI3-K target, Akt1. Both AG1478 and anti-EGFr inhibited H2O2-induced EGFr activation indicating that H2O2 can activate EGFr via EGFr ligand release. Tyrphostin AG1478 also had no effect on H2O2 inhibition of CCh-stimulated Cl- secretion across T84 monolayers. In summary, H2O2 activates EGFr in colonic epithelial cells, probably via the release of an EGFr ligand, but this effect does not account for inhibition of Ca2+-dependent Cl− secretion by H2O2. Because we have previously shown that EGFr activated by other means can lead to inhibition of Cl− secretion, our data imply selective outcomes of EGFr signaling depending on its mode of activation. Supported by the Crohn's and Colitis Foundation of America and NIH.

Chemokines induce matrix metalloproteinase-2 through activation of epidermal growth factor receptor in arterial smooth muscle cells

Matrix metalloproteinases (MMP) are critical to smooth muscle cell (SMC) migration in vivo. MMP-2 dysregulation has been implicated in the pathogenesis of abnormal arterial remodeling, aneurysm formation, and atherosclerotic plaque structure and stability. The chemokine receptors CCR3 and CXCR4 are present and functional on SMC and are up-regulated in vascular diseases such as atherosclerosis. We sought to determine a potential mechanism for chemokine receptor-mediated effects on the vasculature by asking whether the chemokines eotaxin (CCL11), the ligand for CCR3, and stromal cell-derived cell factor (SDF-1, CXCL12), the ligand for CXCR4, induce MMP-2 in SMC. Studies were then performed to define the signaling pathways involved. As determined by RT-PCR, Western blotting and zymography, SDF-1 and eotaxin induce MMP-2 mRNA, protein, and activity in SMC. An anti-CCR3 antibody and a CXCR4 antagonist blocked proMMP-2 induction by SDF-1 and eotaxin, the respective ligands for the chemokine receptors CXCR4 and CCR3, suggesting that the inductions by these chemokines are receptor-mediated. Receptor cross-talk between G-protein-coupled receptors (GPCR) and the epidermal growth factor receptor (EGFR) is a method of expanding the GPCRs' signaling repertoire. We demonstrate, for the first time to our knowledge, that in SMC, chemokine induction of proMMP-2 is dependent on activation of the EGFR. Interestingly, by blocking the ligand binding domain of EGFR, we demonstrate that activation of EGFR by SDF-1 and eotaxin occurs through different cellular pathways. The pro-inflammatory chemokines eotaxin and SDF induce proMMP-2 activation of EGFR through two different pathways. SDF and eotaxin, as regulators of proMMP-2 expression and by engaging in receptor cross-talk, may play critical roles in atherosclerosis, restenosis, and plaque rupture. These ligands and their respective receptors, CXCR4 and CCR3, therefore may serve as future potential therapeutic targets.

The epidermal growth factor receptor as a target for gastrointestinal cancer therapy.

The epidermal growth factor receptor (EGFR) is a member of the family of transmembrane protein kinase receptors known as the erbB or HER receptor family. When activated, EGFR phosphorylates and activates other intracellular proteins that affect cell signaling pathways, cellular proliferation, control of apoptosis and angiogenesis. EGFR signaling is best thought of as a network of activating and inactivating proteins with EGFR as the entry point into the network. EGFR overexpression occurs in most GI malignancies and while data are not entirely consistent, EGFR overexpression often confers a poor prognosis in those GI malignancies that have been studied. It often correlates with poorly differentiated histology, more advanced stage and other known poor prognostic markers. The EGFR is a tempting target because of its presence and overexpression on so many tumor types. However, downstream of the EGFR are several proteins that may be activated without EGFR thus allowing blockade to be overcome. Therefore, while blocking the activity of the EGFR protein appears to be a promising anticancer strategy, a simplistic strategy of blocking only EGFR is likely to only impact a minority of patients. It is time for the laboratory and clinical researchers to work closely together to develop this treatment strategy, moving back and forth from clinical to laboratory to best understand how to block this network effectively enough to produce a broader antitumor effect. While multiple methods of targeting the EGFR pathway are under development, including the inhibition of downstream proteins, only two modalities have entered clinical trials in GI malignancies: small molecule inhibitors of the intracellular kinase domain of EGFR and antibodies designed to block the extracellular ligand-binding domain of EGFR. EGFR inhibitors are still experimental in every GI malignancy with the notable exception of cetuximab that is approved for second or third-line therapy of metastatic colorectal cancer, used either alone or in combination with irinotecan (Camptosar, Kalamazoo, Mich). Data on clinical applications of these agents in GI malignancies will be the focus of this paper.

Activation of Proteinase-Activated Receptor 1 Promotes Human Colon Cancer Cell Proliferation Through Epidermal Growth Factor Receptor Transactivation

Serine proteases are now considered as crucial contributors to the development of human colon cancer. We have shown recently that thrombin is a potent growth factor for colon cancer cells through activation of the aberrantly expressed protease-activated receptor 1 (PAR1). Here, we analyzed the signaling pathways downstream of PAR1 activation, which lead to colon cancer cell proliferation in HT-29 cells. Our data are consistent with the following cascade of events on activation of PAR1 by thrombin or specific activating peptide: (a) a matrix metalloproteinase-dependent release of transforming growth factor-alpha (TGF-alpha) as shown with TGF-alpha blocking antibodies and measurement of TGF-alpha in culture medium; (b) TGF-alpha-mediated activation of epidermal growth factor receptor (EGFR) and subsequent EGFR phosphorylation; and (c) activation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and subsequent cell proliferation. The links between these events are shown by the fact that stimulation of cell proliferation and ERK1/2 on activation of PAR1 is reversed by the MMP inhibitor batimastat, TGF-alpha neutralizing antibodies, EGFR ligand binding domain blocking antibodies, and the EGFR tyrosine kinase inhibitors AG1478 and PD168393. Therefore, transactivation of EGFR seems to be a major mechanism whereby activation of PAR1 results in colon cancer cell growth. Finally, PAR1 activation induces Src phosphorylation, which is reversed by using the Src tyrosine kinase inhibitor PP2, suggesting that Src activation plays a permissive role for PAR1-mediated ERK1/2 activation and cell proliferation probably acting downstream of the EGFR. These data explain how thrombin exerts robust trophic action on colon cancer cells and underline the critical role of EGFR transactivation.

Transactivation of the Epidermal Growth Factor Receptor in Colonic Epithelial Cells by Carbachol Requires Extracellular Release of Transforming Growth Factor-α

We have shown previously that the muscarinic agonist, carbachol (CCh), transactivates the epidermal growth factor receptor (EGFr) via calmodulin, Pyk-2, and Src kinase activation. EGFr phosphorylation causes extracellular signal-regulated kinase (ERK) activation and inhibits CCh-stimulated chloride secretion across intestinal epithelial cells. Here we investigated whether CCh-stimulated EGFr transactivation involves EGFr ligand release. Pre-incubation of T(84) cell monolayers with a neutralizing antibody to the EGFr ligand binding domain decreased CCh-induced phosphorylation of EGFr and ERK. CCh-stimulated efflux of (86)Rb+ from T(84) cell monolayers, which parallels changes in chloride secretion, was potentiated by anti-EGFr pre-incubation. Anti-EGFr did not reduce CCh-stimulated Pyk-2 phosphorylation. Co-incubation with the Src kinase inhibitor PP2 and anti-EGFr had an additive inhibitory effect on CCh-induced ERK phosphorylation greater than either inhibitor alone. CCh caused the basolateral release of transforming growth factor alpha (TGF-alpha) into T(84) cell bathing media. A metalloproteinase inhibitor, WAY171318, reduced CCh-induced phosphorylation of ERK and completely blocked EGFr phosphorylation and TGF-alpha release. We conclude that CCh-stimulated EGFr transactivation and subsequent ERK activation, a pathway that limits CCh-induced chloride secretion, is mediated by metalloproteinase-dependent extracellular release of TGF-alpha and intracellular Src activation. These findings have important implications for our understanding of the role of growth factors in regulating epithelial ion secretion.

Modulation of the Kv1.3 potassium channel by receptor tyrosine kinases.

The voltage-dependent potassium channel, Kv1.3, is modulated by the epidermal growth factor receptor (EGFr) and the insulin receptor tyrosine kinases. When the EGFr and Kv1.3 are coexpressed in HEK 293 cells, acute treatment of the cells with EGF during a patch recording can suppress the Kv1.3 current within tens of minutes. This effect appears to be due to tyrosine phosphorylation of the channel, as it is blocked by treatment with the tyrosine kinase inhibitor erbstatin, or by mutation of the tyrosine at channel amino acid position 479 to phenylalanine. Previous work has shown that there is a large increase in the tyrosine phosphorylation of Kv1.3 when it is coexpressed with the EGFr. Pretreatment of EGFr and Kv1.3 cotransfected cells with EGF before patch recording also results in a decrease in peak Kv1.3 current. Furthermore, pretreatment of cotransfected cells with an antibody to the EGFr ligand binding domain (alpha-EGFr), which blocks receptor dimerization and tyrosine kinase activation, blocks the EGFr-mediated suppression of Kv1.3 current. Insulin treatment during patch recording also causes an inhibition of Kv1.3 current after tens of minutes, while pretreatment for 18 h produces almost total suppression of current. In addition to depressing peak Kv1.3 current, EGF treatment produces a speeding of C-type inactivation, while pretreatment with the alpha-EGFr slows C-type inactivation. In contrast, insulin does not influence C-type inactivation kinetics. Mutational analysis indicates that the EGF-induced modulation of the inactivation rate occurs by a mechanism different from that of the EGF-induced decrease in peak current. Thus, receptor tyrosine kinases differentially modulate the current magnitude and kinetics of a voltage-dependent potassium channel.

Inhibition of epidermal growth factor receptor-associated tyrosine kinase blocks glioblastoma invasion of the brain.

Glioblastoma multiforme is a malignant primary brain tumor associated with short patient survival despite aggressive treatment, in part because of its propensity to aggressively infiltrate into brain tissue. Glioblastoma multiforme is also unique because it is the only nonepithelial human tumor for which excessive activation of epidermal growth factor receptor (EGFR) has been consistently linked to tumor growth and patient survival, and EGFR activation promotes glioblastoma multiforme infiltration in vitro. Cocultures of human glioblastoma spheroids (derived from three separate patients) and fetal rat brain aggregates were examined for infiltration using confocal microscopy, in the presence of 0 to 100 mumol/L genistein, a tyrosine kinase (TK) inhibitor, and 3 mumol/L tyrphostin A25, a specific EGFR-TK inhibitor. Infiltration (not attachment) was completely inhibited by genistein at 10 mumol/L, the IC20 for monolayer growth inhibition in two cell lines. Tyrphostin A25 at 3 mumol/L (the IC20 for monolayers) reduced invasion in a third cell line from 38.8 +/- 6.1% invasion-hour per hour (n = 5) to 2.9 +/- 1.2% invasion-hour per hour (n = 6) (P = 0.0002, two-tailed t test, 93% inhibition), and from 0.54 +/- 0.065% per hour (slope) to 0.028 +/- 0.018% per hour (P = 0.00001, 95% inhibition). Maximal percent invasion was reduced from 100 +/- 0 to 7.4 +/- 5.6% of the fetal rat brain aggregate. No change was detected in EGFR-associated tyrosine phosphorylation at those doses in monolayers by 32P immunolabeling, consistent with the known effects of low concentrations of TK inhibitors. An increase in expression of wild-type and truncated EGFR was demonstrated by Western blotting. Invasion was equally well inhibited by a monoclonal antibody to the high-affinity ligand binding domain of EGFR and not by antibody to an inactive domain. Our observations support the role of EGFR activation as a determinant by which glioblastoma invades normal brain tissue, and we show that invasion can be effectively inhibited at much lower concentrations of TK inhibitors than are necessary for growth suppression.

Inhibition of Epidermal Growth Factor Receptor-associated Tyrosine Kinase Blocks Glioblastoma Invasion of the Brain<subtitle>Experimental Studies</subtitle>

OBJECTIVE : Glioblastoma multiforme is a malignant primary brain tumor associated with short patient survival despite aggressive treatment, in part because of its propensity to aggressively infiltrate into brain tissue. Glioblastoma multiforme is also unique because it is the only nonepithelial human tumor for which excessive activation of epidermal growth factor receptor (EGFR) has been consistently linked to tumor growth and patient survival, and EGFR activation promotes glioblastoma multiforme infiltration in vitro. METHODS : Cocultures of human glioblastoma spheroids (derived from three separate patients) and fetal rat brain aggregates were examined for infiltration using confocal microscopy, in the presence of 0 to 100 nmol/L genistein, a tyrosine kinase (TK) inhibitor, and 3 nmol/L tyrphostin A25, a specific EGFR-TK inhibitor. RESULTS : Infiltration (not attachment) was completely inhibited by genistein at 10 nmol/L, the IC 20 for monolayer growth inhibition in two cell lines. Tyrphostin A25 at 3 nmol/L (the IC 20 for monolayers) reduced invasion in a third cell line from 38.8 ± 6.1% invasion-hour per hour (n = 5) to 2.9 ± 1.2% invasion-hour per hour (n = 6) (P = 0.0002, two-tailed t test, 93% inhibition), and from 0.54 ± 0.065% per hour (slope) to 0.028 ± 0.018% per hour (P = 0.00001, 95% inhibition). Maximal percent invasion was reduced from 100 ± 0 to 7.4 ± 5.6% of the fetal rat brain aggregate. No change was detected in EGFR-associated tyrosine phosphorylation at those doses in monolayers by 32 P immunolabeling, consistent with the known effects of low concentrations of TK inhibitors. An increase in expression of wild-type and truncated EGFR was demonstrated by Western blotting. Invasion was equally well inhibited by a monoclonal antibody to the high-affinity ligand binding domain of EGFR and not by antibody to an inactive domain. CONCLUSION : Our observations support the role of EGFR activation as a determinant by which glioblastoma invades normal brain tissue, and we show that invasion can be effectively inhibited at much lower concentrations of TK inhibitors than are necessary for growth suppression.

Effect of carboxyl terminal truncation on the tyrosine kinase activity of the epidermal growth factor receptor

The carboxyl terminal domain of the epidermal growth factor receptor (EGFR) is an important regulatory region in mediating the tyrosine kinase-dependent biological effects of EGF. The effect of a 164-amino-acid carboxyl deletion of the EGFR or the EGFR cytoplasmic kinase domain on in vitro tyrosine kinase activity was assessed. C′-terminal truncation of the EGFR resulted in dependence on Mn 2+ for full activity. The EGFR kinase domain (kd EGFR) and the C′-terminally truncated kinase domain (kd c′1022 EGFR) also exhibited a strong preference for Mn 2+ compared to Mg 2+, with kd c′1022 EGFR being completely inactive in the presence of Mg 2+ alone. Sphingosine or ammonium sulfate specifically activated both kd EGFR and kd c′1022 EGFR. EGFR and c′1022 EGFR displayed similar EGF-stimulated in vitro tyrosine kinase activities; however, kd EGFR was 5- to 10-fold more active in vitro than kd c′1022 EGFR. Thus, the regulatory contribution of the C′-terminus is most evident when the EGFR ligand binding domain is removed. These results indicate that an intact EGFR C′-terminus is necessary for the protein to assume a fully active conformation.