Epidermal Growth Factor Receptor ErbB1 Research Articles

In silico exploration of anti-prostate cancer compounds from differential expressed genes

Prostate cancer (PCa) is a complex and biologically diverse disease with no curative treatment options at present. This study aims to utilize computational methods to explore potential anti-PCa compounds based on differentially expressed genes (DEGs), with the goal of identifying novel therapeutic indications or repurposing existing drugs. The methods employed in this study include DEGs-to-drug prediction, pharmacokinetics prediction, target prediction, network analysis, and molecular docking. The findings revealed a total of 79 upregulated DEGs and 110 downregulated DEGs in PCa, which were used to identify drug compounds capable of reversing the dysregulated conditions (dexverapamil, emetine, parthenolide, dobutamine, terfenadine, pimozide, mefloquine, ellipticine, and trifluoperazine) at a threshold probability of 20% on several molecular targets, such as serotonin receptors 2a/2b/2c, HERG protein, adrenergic receptors alpha-1a/2a, dopamine D3 receptor, inducible nitric oxide synthase (iNOS), epidermal growth factor receptor erbB1 (EGFR), tyrosine-protein kinases, and C-C chemokine receptor type 5 (CCR5). Molecular docking analysis revealed that terfenadine binding to inducible nitric oxide synthase (-7.833 kcal.mol−1) and pimozide binding to HERG (-7.636 kcal.mol−1). Overall, binding energy ΔGbind (Total) at 0 ns was lower than that of 100 ns for both the Terfenadine-iNOS complex (-101.707 to -103.302 kcal.mol−1) and Ellipticine-TOPIIα complex (-42.229 to -58.780 kcal.mol−1). In conclusion, this study provides insight on molecular targets that could possibly contribute to the molecular mechanisms underlying PCa. Further preclinical and clinical studies are required to validate the therapeutic effectiveness of these identified drugs in PCa disease.

Virtual Screening of Different Subclasses of Lignans with Anticancer Potential and Based on Genetic Profile.

Cancer is a multifactorial disease that continues to increase. Lignans are known to be important anticancer agents. However, due to the structural diversity of lignans, it is difficult to associate anticancer activity with a particular subclass. Therefore, the present study sought to evaluate the association of lignan subclasses with antitumor activity, considering the genetic profile of the variants of the selected targets. To do so, predictive models were built against the targets tyrosine-protein kinase ABL (ABL), epidermal growth factor receptor erbB1 (EGFR), histone deacetylase (HDAC), serine/threonine-protein kinase mTOR (mTOR) and poly [ADP-ribose] polymerase-1 (PARP1). Then, single nucleotide polymorphisms were mapped, target mutations were designed, and molecular docking was performed with the lignans with the best predicted biological activity. The results showed more anticancer activity in the dibenzocyclooctadiene, furofuran and aryltetralin subclasses. The lignans with the best predictive values of biological activity showed varying binding energy results in the presence of certain genetic variants.

Plexins as Regulators of Cancer Cell Proliferation, Migration, and Invasivity.

Plexins are a family of nine single-pass transmembrane receptors with a conserved GTPase activating protein (GAP) domain. The plexin family is divided into four subfamilies: Type-A, type-B, type-C, and type-D plexins. Plexins function as receptors for axon guidance factors of the semaphorin family. The semaphorin gene family contains 22 genes that are divided into eight subclasses of which subclasses three to seven represent vertebrate semaphorins. The plexins and their semaphorin ligands have important roles as regulators of angiogenesis, cancer proliferation, and metastasis. Class 3 semaphorins, with the exception of sema3E, are the only semaphorins that do not bind directly to plexins. In order to transduce their signals, they bind instead to complexes consisting of receptors of the neuropilin family and various plexins. Some plexins also form complexes with tyrosine-kinase receptors such as the epidermal growth factor receptor ErbB2, the mesenchymal epithelial transition factor receptor (MET), and the Vascular endothelial growth factor receptor 2 (VEGFR2) and, as a result, can modulate cell proliferation and tumor progression. This review focuses on the roles of the different plexins in the control of cancer cell proliferation and invasiveness. Plexins also affect tumor progression and tumor metastasis by indirect mechanisms, such as modulation of angiogenesis and immune responses. However, these topics are not covered in the present review.

StackCPA: A stacking model for compound-protein binding affinity prediction based on pocket multi-scale features

Accurately predicting compound-protein binding affinity is a crucial task in drug discovery. Computational models offer the advantages of short time, low cost and safety compared to traditional drug development. Pocket is the key binding region of the protein, which provides invaluable information for drug repositioning and drug design. In this study, we propose an ensemble learning model, called StackCPA, to predict the compound-protein binding affinity. The model integrates multi-scale features of protein pocket and compound through a transfer learning strategy. The protein pocket is described in a fine-grained way by atomic level, residue level and subdomain level. The proposed model StackCPA is evaluated on three binding affinity benchmark datasets. The experiment results show that StackCPA achieves the best performance on all the three datasets in comparison with other state-of-the-art deep learning models. The ablation study shows that the protein pocket can provide sufficient information for affinity prediction and its multi-scale features enable the model to further improve the prediction performance. In addition, the case study for epidermal growth factor receptor erbB1 (EGFR) indicates that StackCPA could serve as an effective tool for drug repurposing. Source codes and data of StackCPA are available at https://github.com/CSUBioGroup/StackCPA.

Protease signaling regulates apical cell extrusion, cell contacts, and proliferation in epithelia.

Mechanisms that sense and regulate epithelial morphogenesis, integrity, and homeostasis are incompletely understood. Protease-activated receptor 2 (Par2), the Par2-activating membrane-tethered protease matriptase, and its inhibitor, hepatocyte activator inhibitor 1 (Hai1), are coexpressed in most epithelia and may make up a local signaling system that regulates epithelial behavior. We explored the role of Par2b in matriptase-dependent skin abnormalities in Hai1a-deficient zebrafish embryos. We show an unexpected role for Par2b in regulation of epithelial apical cell extrusion, roles in regulating proliferation that were opposite in distinct but adjacent epithelial monolayers, and roles in regulating cell-cell junctions, mobility, survival, and expression of genes involved in tissue remodeling and inflammation. The epidermal growth factor receptor Erbb2 and matrix metalloproteinases, the latter induced by Par2b, may contribute to some matriptase- and Par2b-dependent phenotypes and be permissive for others. Our results suggest that local protease-activated receptor signaling can coordinate cell behaviors known to contribute to epithelial morphogenesis and homeostasis.

Graphene Liquid Enclosure for Single-Molecule Analysis of Membrane Proteins in Whole Cells Using Electron Microscopy.

Membrane proteins govern many important functions in cells via dynamic oligomerization into active complexes. However, analytical methods to study their distribution and functional state in relation to the cellular structure are currently limited. Here, we introduce a technique for studying single-membrane proteins within their native context of the intact plasma membrane. SKBR3 breast cancer cells were grown on silicon microchips with thin silicon nitride windows. The cells were fixed, and the epidermal growth factor receptor ErbB2 was specifically labeled with quantum dot (QD) nanoparticles. For correlative fluorescence- and liquid-phase electron microscopy, we enclosed the liquid samples by chemical vapor deposited (CVD) graphene films. Depending on the local cell thickness, QD labels were imaged with a spatial resolution of 2 nm at a low electron dose. The distribution and stoichiometric assembly of ErbB2 receptors were determined at several different cellular locations, including tunneling nanotubes, where we found higher levels of homodimerization at the connecting sites. This experimental approach is applicable to a wide range of cell lines and membrane proteins and particularly suitable for studies involving both inter- and intracellular heterogeneity in protein distribution and expression.

Actin binding GFP allows 4D in vivo imaging of myofilament dynamics in the zebrafish heart and the identification of Erbb2 signaling as a remodeling factor of myofibril architecture.

Dilated cardiomyopathy is a leading cause of congestive heart failure and a debilitating complication of antineoplastic therapies. Despite disparate causes for dilated cardiomyopathy, maladaptive cardiac remodeling and decreased systolic function are common clinical consequences, begging an investigation of in vivo contractile dynamics in development and disease, one that has been impossible to date. To image myocardial contractile filament dynamics in vivo and to assess potential causes of dilated cardiomyopathy in antineoplastic therapies targeting the epidermal growth factor receptor Erbb2. We generated a transgenic zebrafish line expressing an actin-binding green fluorescent protein in cardiomyocytes, allowing an in vivo imaging of myofilaments. Analysis of this line revealed architectural differences in myofibrils of the distinct cardiomyocyte subtypes. We used this model to investigate the effects of Erbb2 signaling on myofibrillar organization because drugs targeting ERBB2 (HER2/NEU) signaling, a mainstay of breast cancer chemotherapy, cause dilated cardiomyopathy in many patients. High-resolution in vivo imaging revealed that Erbb2 signaling regulates a switch between a dense apical network of filamentous myofibrils and the assembly of basally localized myofibrils in ventricular cardiomyocytes. Using this novel line, we compiled a reference for myofibrillar microarchitecture among myocardial subtypes in vivo and at different developmental stages, establishing this model as a tool to analyze in vivo cardiomyocyte contractility and remodeling for a broad range of cardiovascular questions. Furthermore, we applied this model to study Erbb2 signaling in cardiomyopathy. We show a direct link between Erbb2 activity and remodeling of myofibrils, revealing an unexpected mechanism with potentially important implications for prevention and treatment of cardiomyopathy.

Transmembrane Domain Targeting Peptide Antagonizing ErbB2/Neu Inhibits Breast Tumor Growth and Metastasis

Breast cancer is still a deadly disease despite major achievements in targeted therapies designed to block ligands or ligand-binding subunits of major tyrosine kinase receptors. Relapse is significant and metastases deleterious, which demands novel strategies for fighting this disease. Here, we report a proof-of-concept experiment demonstrating that small peptides interfering with the transmembrane domain of the tyrosine kinase epidermal growth factor receptor ErbB2 exhibit anticancer properties when used at micromolar dosages in a genetically engineered mouse model of breast cancer. Different assays demonstrate the specificity of the ErbB2-targeting peptide, which induces long-term reduction of ErbB2 phosphorylation and Akt signaling consistent with reduced tumor cell proliferation and increased survival. Microcomputed tomography analysis established the antimetastatic activity of the peptide and its impact on primary tumor growth. This reveals the interior of the cell membrane as an unexplored dimension for drug design.

Pertuzumab counteracts the inhibitory effect of ErbB2 on degradation of ErbB3

Overexpression of ErbB2 and ErbB3 is found in several human cancers, and ErbB2-ErbB3 heterodimers are known as the most potent signaling units among ErbB dimers. While ErbB2 probably undergoes weak endocytosis, ErbB3 is readily internalized even in the absence of added ligand and without requirement for kinase activity. Overexpression of ErbB2 has been demonstrated to inhibit epidermal growth factor-induced internalization and degradation of epidermal growth factor receptor. This happens due to epidermal growth factor receptor-ErbB2 dimerization and can be counteracted by the anti-ErbB2 antibody pertuzumab, which binds the dimerization arm of ErbB2. Pertuzumab does also inhibit ErbB2-ErbB3 dimerization, but to what extent this has effect on constitutive and/or ligand-induced downregulation of ErbB3 is not known. In this study, we demonstrate that expression of ErbB2 as such did not block constitutive internalization of ErbB3, but that heregulin-induced degradation of ErbB3 was significantly slowed in cells expressing high levels of ErbB2. Incubation with pertuzumab did, however, counteract this effect. This indicates that the formation of ErbB2-ErbB3 heterodimers inhibits downregulation of ErbB3 and supports the notion that pertuzumab inhibits ErbB2 dimerization. The inhibitory effect of pertuzumab on ligand-induced ErbB2-ErbB3 heterodimerization was confirmed by the observation that pertuzumab inhibited heregulin-induced phosphorylation of ErbB3 in cells expressing ErbB2 and efficiently reduced heregulin-induced downstream signaling in cells expressing low levels of ErbB2. Altogether the results indicate that pertuzumab can be a valuable therapeutic agent not only in cancers overexpressing ErbB2 but also in cancers co-expressing ErbB2 and ErbB3.

ErbB1 epidermal growth factor receptor is a valid target for reducing the effects of multiple inhibitors of axonal regeneration

Pharmacological inhibitors of epidermal growth factor receptor (ErbB1) attenuate the ability of CNS myelin to inhibit axonal regeneration. However, it has been claimed that such effects are mediated by off-target interactions. We have tested the role of ErbB1 in axonal regeneration by culturing neurons from ErbB1 knockout mice in the presence of various inhibitors of axonal regeneration: CNS myelin, chondroitin sulfate proteoglycans (CSPG), fibrinogen or polyinosinic:polycytidylic acid (poly I:C). We confirmed that ErbB1 was activated in cultures of cerebellar granule cells exposed to inhibitors of axonal regeneration and that ErbB1 kinase inhibitors promoted neurite outgrowth under these conditions. In the presence of myelin, fibrinogen, CSPG and poly I:C ErbB1 −/− neurons grew longer neurites than neurons expressing ErbB1. Furthermore, inhibitors of ErbB1 kinase did not improve neurite outgrowth from ErbB1 −/− neurons, ruling out an off-target mechanism of action. ErbB1 kinase activity is therefore a valid target for promoting axonal elongation in the presence of many of the molecules believed to contribute to the failure of axonal regeneration in the injured CNS.

Quantification of differential ErbB1 and ErbB2 cell surface expression and spatial nanoclustering through plasmon coupling.

Cell surface receptors play ubiquitous roles in cell signaling and communication and their expression levels are important biomarkers for many diseases. Expression levels are, however, only one factor that determines the physiological activity of a receptor. For some surface receptors, their distribution on the cell surface, especially their clustering, provides additional mechanisms for regulation. To access this spatial information robust assays are required that provide detailed insight into the organization of cell surface receptors on nanometer length scales. In this manuscript, we demonstrate through combination of scattering spectroscopy, electron microscopy, and generalized multiple particle Mie theory (GMT) simulations that the density- and morphology-dependent spectral response of Au nanoparticle (NP) immunolabels bound to the epidermal growth factor receptors ErbB1 and ErbB2 encodes quantitative information of both the cell surface expression and spatial clustering of the two receptors in different unliganded in vitro cancer cell lines (SKBR3, MCF7, A431). A systematic characterization of the collective spectral responses of NPs targeted at ErbB1 and ErbB2 at various NP concentrations indicates differences in the large-scale organization of ErbB1 and ErbB2 in cell lines that overexpress these receptors. Validation experiments in the scanning electron microscope (SEM) confirm that NPs targeted at ErbB1 on A431 are more strongly clustered than NPs bound to ErbB2 on SKBR3 or MCF7 at overall comparable NP surface densities. This finding is consistent with the existence of larger receptor clusters for ErbB1 than for ErbB2 in the plasma membranes of the respective cells.

PD01-09: Identifying Novel Mechanisms of Resistance to Lapatinib in ERBB2+ Breast Cancer Cells through Whole Genome Mutational Analysis.

Abstract Background: Overexpression of the epidermal growth factor receptor ERBB2 (HER2) is found in 20% of human breast cancers. Therapies targeting ERBB2 including trastuzumab and lapatinib have significantly improved the outlook for women with ERBB2+ breast cancer. However, resistance to these agents occurs frequently and remains a significant clinical problem. In the case of lapatinib resistance, the mechanism(s) of resistance remain poorly understood, since the current proposed rationale thought to limit lapatinib's anti-tumor effects has been difficult to reconcile with clinical data. Therefore, we hypothesize that novel mechanisms of resistance could be identified by mapping genomic variations in ERBB2+ cells with acquired resistance to lapatinib. The identification of such mutations may provide insights into mechanisms of resistance and may indicate therapeutic strategies to overcome lapatinib resistance in ERBB2+ breast cancer. Material and Methods: SKBR3 breast cancer cells resistant to lapatinib were generated through serial passage by exposure of drug sensitive parental SKBR3 cells to increasing concentrations of lapatinib up to the peak plasma concentration observed in human subjects (2.6 uM (SK-lapR)). Multiple signaling pathways in lapatinib sensitive and resistant cells were interrogated by Reverse Phase Protein Array (RPPA) and western blot analysis. To identify genome wide somatic mutations, the Exome of lapatinib resistant and sensitive SKBR3 cells was sequenced utilizing next generation deep sequencing. Following exclusion of germline variants, the acquired gene mutations in lapatinib resistant SKBR3 cells were confirmed by DNA re-sequencing of PCR amplified DNA segments. Results and Discussion: Analysis of activated signaling pathways in lapatinib resistant and sensitive SKBR3 cells did not confirm any of the previously proposed mechanisms of resistance. In particular, these cells show no activation of AKT or alternative receptor tyrosine kinases such as IGF-IR, ERBB3 or c-Met. However they exhibit sustained activation of mTORC1 and ERK1/2, as well as phosphorylation of STAT3, STAT5, rpS6 and CREB. Initial sequence analysis of exome and transcriptome reveals the presence of 76 single nucleotide variants/Indels differing between sensitive and resistant cells with 34/76 validated as true mutations present in the genome of lapatinib resistant SKBR3 cells, including mutations in LATS2, MAP3K5, SMAD3 and PDGFRA. This is the first exome sequence analysis to be reported which defines a drug resistant phenotype in ERBB2+ breast cancer. Ongoing work includes investigation of mutations as drug resistance mediators and analysis of copy number variations and gene fusions/translocations to systematically search for molecular alterations, with the goal of providing a rationale for the design of new combination therapies aimed at lapatinib resistance for ERBB2+ breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD01-09.

HER2 Codon 655 G-Allele Is Associated With Reductions in Plasma High-Density Lipoprotein Levels in Breast Cancer Patients Treated With Tamoxifen

BackgroundRetrospective studies have revealed that overexpression of the epidermal growth factor receptor ErbB-2 (HER2) reduced the efficacy of tamoxifen therapy, which is associated with an increased risk for cardiovascular events....

Abstract C58: Humanization and characterization of an anti-ErbB-3 murine monoclonal antibody.

Abstract Background: The signaling network downstream of the human epidermal growth factor receptors ErbB-1 and ErbB-2 have been extensively targeted by cancer therapeutics. Although initially efficacious in a subset of patients, drugs targeting these receptors led invariably to resistance which is often associated with reactivation of the ErbB-3/PI3K-Akt axis. This may be overcome by ErbB-3 ligands that abrogates receptor-mediated signaling. Methods: the murine MP-RM-1 monoclonal antibody that specifically binds ErbB-3 ectodomain was chimerized by replacing the murine constant regions with the human constant regions and humanized by gratfing Complementarity Determining Regions from the murine into a human antibody framework using a computer assisted molecular modelling. Recombinant genes were placed into the pCDNA3.1 expression vector and transfected into Chinese Hamster Ovary-S cells. Antibodies were screened on the basis of their ability to inhibit Neuregulin-1 (NRG-1)-induced activation of the ErbB-3/PI3K-Akt axis in IR-8 human melanoma cells (Table 1). Four antibody variants, i.e., cMP-RM-1 #1, hMP-RM-1 #6, hMP-RM-1 #10, and hMP-RM-1 #20, were selected and further tested for their ability to promote ErbB-3 downregulation and to inhibit in vitro and in vivo growth of tumor cells. Results: Four chimeric (c) and sixteen humanized (h) antibody variants were obtained. All variants showed to possess antigen binding affinity comparable to that of their parental antibody. Initial screening identified one chimeric (cMP-RM-1 #1) and three humanized (hMP-RM-1 #6, hMP-RM-1 #10, hMP-RM-1 #20) antibodies as the most effective in inhibiting ErbB-3/Akt phosphorylation and promotion of ErbB-3 downregulation. Further testing revealed the antibodies potently inhibited colony formation ability of tumor cells and halted growth of melanoma and ovarian cancer xenografts in nude mice. Treatment with the antibodies was associated with a decreased ErbB-3 and Akt phosphorylation and ErbB-3 expression in the excised tumor tissue. Conclusions: This successful production of chimeric/humanized antibodies possessing antitumor activity in vitro and in vivo may provide novel candidates for ErbB-3-targeted therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C58.

A Single-Arm Phase II Trial of First-Line Paclitaxel in Combination with Lapatinib in HER2-Overexpressing Metastatic Breast Cancer

Introduction: Lapatinib, an orally active tyrosine kinase inhibitor of epidermal growth factor receptor ErbB1 (EGFR) and ErbB2 (HER2), has activity as monotherapy and in combination with chemotherapy in HER2-overexpressing metastatic breast cancer (MBC). Methods: This phase II single-arm trial assessed the safety and efficacy of first-line lapatinib in combination with paclitaxel in previously untreated patients with HER2-overexpressing MBC. The primary endpoint was the overall response rate (ORR). Secondary endpoints were the duration of response (DoR), time to response, time to progression, progression-free survival (PFS), overall survival, and the incidence and severity of adverse events. All endpoints were investigator- and independent review committee (IRC)-assessed. Results: The IRC-assessed ORR was 51% (29/57 patients with complete or partial response) while the investigator-assessed ORR was 77% (44/57). As per the IRC, the median DoR was 39.7 weeks, and the median PFS was 47.9 weeks. The most common toxicities were diarrhea (56%), neutropenia (44%), rash (40%), fatigue (25%), and peripheral sensory neuropathy (25%). Conclusions: First-line lapatinib plus paclitaxel for HER2-overexpressing MBC produced an encouraging ORR with manageable toxicities. This combination may be useful in first-line treatment for patients with HER2-overexpressing MBC and supports the ongoing evaluation of this combination as first-line therapy in HER2-overexpressing MBC.

Protein-tyrosine Phosphatase PTPN9 Negatively Regulates ErbB2 and Epidermal Growth Factor Receptor Signaling in Breast Cancer Cells

ErbB family of the receptor protein-tyrosine kinase plays an important role in the progression of human cancers including breast cancer. Finding protein-tyrosine phosphatase (PTPs) that can specifically regulate the function of ErbB should help design novel therapies for treatment. By performing a small interfering RNA screen against 43 human PTPs, we find that knockdown of protein-tyrosine phosphatase PTPN9 significantly increases ErbB2 tyrosyl phosphorylation in the SKBR3 breast cancer cell line. In addition, knockdown of PTPN9 expression also enhances tyrosyl phosphorylation of the ErbB1/epidermal growth factor receptor (EGFR) in the MDA-MB-231 breast cancer cell line. Conversely, increasing expression of PTPN9 wild type (WT) inhibits tyrosyl phosphorylation of ErbB2 and EGFR. To test whether ErbB2 and EGFR are substrates of PTPN9, PTPN9 WT, and a substrate trapping mutant (PTPN9 DA) are overexpressed in SKBR3 and MDA-MB-231 cells. Compared with vector control, expression of PTPN9 WT significantly inhibits whereas expression of PTPN9 DA dramatically enhances tyrosyl phosphorylation of ErbB2 and EGFR, respectively. In contrast, expression of PTPN9 WT or DA mutant does not affect tyrosyl phosphorylation of ErbB3 and Shc. Importantly, coimmunoprecipitation and glutathione S-transferase fusion protein pulldown experiments show that tyrosol-phosphorylated ErbB2 or EGFR is preferentially associated with PTPN9 DA compared with PTPN9 WT, indicating that ErbB2 and EGFR are substrates of PTPN9. Furthermore, PTPN9 WT expression specifically impairs EGF-induced STAT3 and STAT5 activation, and inhibits the cell growth in soft agar. Last, PTPN9 WT expression also reduces invasion and MMP2 expression of MDA-MB-231 cells. Our data suggest PTPN9 as a negative regulator of breast cancer cells by targeting ErbB2 and EGFR and inhibiting STAT activation.

Sequence-Dependent Oligomerization of the Neu Transmembrane Domain Suggests Inhibition of “Conformational Switching” by an Oncogenic Mutant

Membrane-spanning epidermal growth factor receptor ErbB2 is of key importance in cell division, in which a dimeric complex of the protein is responsible for tyrosine kinase activation following ligand binding. The rat homologue of this receptor (Neu) is prone to a valine to glutamic acid mutation in the transmembrane domain (TM), resulting in permanent activation and oncogenesis. In this study, the TM domains of Neu and the corresponding oncogenic mutant Neu*, which contains a V to E mutation at position 664 in the TM domain, have been analyzed to improve our understanding of the structural effects of the oncogenic V(664)E mutation. Building on previous work, we have focused here on understanding the sequence dependence of TM helix-helix interactions and any differences in behavior upon introduction of the V(664)E mutation. Using a variety of biochemical and biophysical methods, we find that the rat Neu TM domain forms strong oligomers and, similar to previous observations for the human ErbB2 TM domain, the oncogenic mutation results in a reduced level of self-association. Our data also strongly indicate that the proto-oncogenic Neu TM domain can adopt multiple (at least two) oligomeric conformations in the membrane, possibly corresponding to the active and inactive forms of the receptor, and can "switch" between the two. Further, the oncogenic Neu* mutant appears to inhibit this "conformational switching" of TM dimers, as we observe that dimerization of the Neu* TM domain in the Escherichia coli inner membrane strongly favors a single conformation stabilized by an IXXXV motif (I(659)-XXX-V(663)) originally identified by site-specific infrared spectroscopic studies.

Pertuzumab increases epidermal growth factor receptor down-regulation by counteracting epidermal growth factor receptor-ErbB2 heterodimerization

Epidermal growth factor receptor (EGFR) and ErbB2 readily form heterodimers when both are expressed in the same cell and the EGFR is activated by one of its ligands. Our data show that such heterodimers are constitutively formed also in a ligand-independent manner on overexpression of EGFR and ErbB2 in porcine aortic endothelial cells. Interestingly, cross-linking experiments showed that incubation with the antibody pertuzumab, which has been shown to bind the dimerization arm of ErbB2, resulted in dissolution of EGFR-ErbB2 heterodimers. Incubation with pertuzumab also increased the amount of EGF-induced EGFR homodimers, and under these conditions, endocytosis of radiolabeled EGF was increased. This increase was significant, although slightly more EGF was internalized in cells expressing EGFR only compared with pertuzumab-treated cells expressing both EGFR and ErbB2. By confocal microscopy analysis, more EGF was observed in endosomes on incubation with pertuzumab, and under similar conditions, immunoblotting experiments showed increased EGFR degradation on incubation with both EGF and pertuzumab. These results show that pertuzumab enhanced the endocytic down-regulation of EGFR by counteracting EGFR-ErbB2 heterodimerization. Our previous results showing that ErbB2 counteracts EGFR endocytosis can therefore be explained by tethering of EGFR to ErbB2 at the plasma membrane.

Activation of epidermal growth factor receptor ErbB1 attenuates inhibitory synaptic development in mouse dentate gyrus

Ligands for the epidermal growth factor receptor ErbB1, such as epidermal growth factor (EGF) and transforming growth factor α (TGFα), negatively regulate synaptic maturation of GABAergic neurons in the developing neocortex. Here, we evaluated the effects of these ligands in vivo on developing inhibitory neurons in the dentate gyrus. Hippocampal slices were prepared from postnatal mice repeatedly challenged with EGF or from transgenic mice overexpressing TGFα. We monitored paired pulse depression of field population spikes evoked by perforant path stimulation to estimate the strength of local inhibition. Administration of EGF increased the paired pulse ratio, suggesting a reduction of inhibitory strength. A similar reduction was observed in TGFα transgenic mice. Monitoring miniature and evoked synaptic currents, we estimated EGF effects on synaptic input and output of GABAergic neurons. EGF treatment diminished the amplitude of excitatory postsynaptic currents (EPSCs) in the GABAergic neurons without affecting their miniature EPSCs. EGF also affected output strength of the GABAergic neurons: The frequency of miniature inhibitory postsynaptic currents (IPSCs) and the evoked IPSC/evoked EPSC ratio were decreased in granule cells. In parallel, EGF down-regulated the protein level of vesicular GABA transporter. Thus, ErbB1 ligands influence GABAergic inhibitory synaptic transmission in the developing dentate gyrus.

Matuzumab Short-Term Therapy in Experimental Pancreatic Cancer: Prolonged Antitumor Activity in Combination with Gemcitabine

The epidermal growth factor receptor ErbB-1 is commonly expressed in pancreatic cancer and ErbB-1 targeting has shown promising results. We wanted to evaluate matuzumab (EMD72000), a fully humanized ErbB-1-specific monoclonal antibody in combination with gemcitabine in experimental pancreatic cancer. Using the human pancreatic cancer cell line L3.6pl, we investigated matuzumab in vitro and in vivo. ErbB-1 phosphorylation and downstream pathway activation were evaluated by Western blot. Proliferation and migration assays and fluorescence-activated cell sorting analysis were done. For in vivo studies, we used an orthotopic nude mice model in which 40 mg/kg of matuzumab+/-100 mg/kg of gemcitabine were administered twice weekly. Different treatment durations (7, 14, 21, and 25 days) and varying time points of treatment initiation (days 8, 15, 22, and 29) were evaluated. Ki67, CD31, and phosphorylated p44/42 mitogen-activated protein kinase (MAPK) immunohistochemistry were done. ErbB-1 phosphorylation and downstream MAPK and AKT signaling were significantly reduced by matuzumab. Matuzumab significantly inhibited proliferation and migration in vitro, and induced tumor cell apoptosis in a dose-dependant manner. Matuzumab therapy significantly lowered tumor volume in vivo, reduced lymph node and liver metastases, and decreased microvessel density and tumor cell proliferation. These effects were significantly enhanced when gemcitabine was added. A significant and prolonged antitumor activity was even evident with short-term therapy (7 days) and with a late onset of therapy (day 22 after tumor cell injection). Matuzumab is an effective agent with long-lasting antiproliferative, proapoptotic, antiangiogenic, and antimetastatic activity in human pancreatic cancer models. These effects might be potentiated by gemcitabine.