Significance and role of morphological mechanisms of growth of glial tumors in clinical practice

The two SH2 (Src homology domain 2) domains present in phospholipase C-gamma1 (PLC-gamma1) were assayed for their capacities to recognize the five autophosphorylation sites in the epidermal growth factor receptor. Plasmon resonance and immunological techniques were employed to measure interactions between SH2 fusion proteins and phosphotyrosine-containing peptides. The N-SH2 domain recognized peptides in the order of pY1173 > pY992 > pY1068 > pY1148 >> pY1086, while the C-SH2 domain recognized peptides in the order of pY992 > pY1068 > pY1148 >> pY1086 and pY1173. The major autophosphorylation site, pY1173, was recognized only by the N-SH2 domain. Contributions of the N-SH2 and C-SH2 domains to the association of the intact PLC-gamma1 molecule with the activated epidermal growth factor (EGF) receptor were assessed in vivo. Loss of function mutants of each SH2 domain were produced in a full-length epitope-tagged PLC-gamma1. After expression of the mutants, cells were treated with EGF and association of exogenous PLC-gamma1 with EGF receptors was measured. In this context the N-SH2 is the primary contributor to PLC-gamma1 association with the EGF receptor. The combined results suggest an association mechanism involving the N-SH2 domain and the pY1173 autophosphorylation site as a primary event and the C-SH2 domain and the pY992 autophosphorylation site as a secondary event.

Previous studies have shown that wounding of human corneal epithelial cells (HCECs) results in the release of G-protein-coupled receptor ligands such as ATP and lysophosphatidic acid (LPA), which in turn transactivate epidermal growth factor (EGF) receptor (EGFR) through ectodomain shedding of heparin-binding EGF-like growth factor (HB-EGF). In the present study, the role of extracellular signal-regulated kinases 1/2 (ERK1/2) in regulating EGFR transactivation was investigated. SV40-immortalized HCECs were wounded or stimulated with ATP and LPA. EGFR and ADAM17 activation was analyzed by immunoprecipitation followed by Western blot analysis with phospho-tyrosine or phospho-serine antibodies, respectively. Phosphorylation of ERK and AKT was analyzed by Western blot analysis. HB-EGF shedding was assessed by measuring the release of alkaline phosphatase (AP) in a stably transfected human corneal epithelial (THCE) cell line expressing HB-EGF-AP. ADAM17 and ERK interaction was determined by coimmunoprecipitation. Early, but not late, ERK1/2 phosphorylation in response to wounding, LPA, and ATP was EGFR independent, but sensitive to the inhibitors of calcium influx, protein kinase C and Src kinase. Wounding-, LPA-, and ATP-induced HB-EGF shedding and EGFR activation were attenuated by the MAPK/ERK kinase (MEK) inhibitors PD98059 and U0126, as well as by ADAM10 and -17 inhibitors. ADAM17 was found to be physically associated with active ERK and phosphorylated at serine residues in an ERK-dependent manner in wounded cells. Taken together, our data suggest that in addition to functioning as an EGFR downstream effector, ERK1/2 also mediates ADAM-dependent HB-EGF shedding and subsequent EGFR transactivation in response to a variety of stimuli, including wounding and GPCR ligands.

Porcine epidemic diarrhea virus (PEDV) causes acute and devastating enteric disease in suckling piglets and results in huge economic losses in the pig industry worldwide. To establish productive infection, viruses must first circumvent the host innate immune response. In this study, we found that PEDV infection stimulated epidermal growth factor receptor (EGFR) activation, which has been linked to not only anticancer therapeutics, but also antiviral signaling. Therefore, we determined whether EGFR activation affected PEDV infection by using an activator or overexpression assay. The data showed that EGFR activation enhanced virus replication in both cases. We also found that specific inhibition of EGFR by either inhibitors or small interfering RNA (siRNA) led to a decrease in virus yields. Further analysis revealed that inhibition of EGFR produced augmentation of type I interferon genes. We next observed that the EGFR downstream cascade STAT3 was also activated upon PEDV infection. Similar to the case of EGFR, specific inhibition of STAT3 by either inhibitor or siRNA increased the antiviral activity of interferon and resulted in decreased PEDV RNA levels, and vice versa. The data on STAT3 depletion in combination with EGFR activation suggest that the attenuation of antiviral activity by EGFR activation requires activation of the STAT3 signaling pathway. Taken together, these data demonstrate that PEDV-induced EGFR activation serves as a negative regulator of the type I interferon response and provides a novel therapeutic target for virus infection.IMPORTANCE EGFR is a transmembrane tyrosine receptor that mediates various cellular events, as well as several types of human cancers. In this study, we investigated for the first time the role of EGFR in PEDV infection. We observed that PEDV infection induced EGFR activation. The role of EGFR activation is to impair the antiviral activity of type I interferon, which requires the involvement of the EGFR downstream signaling cascade STAT3. Our findings reveal a new mechanism evolved by PEDV to circumvent the host antiviral response, which might serve as a therapeutic target against virus infection.

In a report accompanying our article, Spigel et al present their findings on combining erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), with onartuzumab (MetMAb; Genentech, South San Francisco, CA), a humanized monovalent monoclonal antibody that blocks ligand activation of MET receptor tyrosine kinase (RTK). MET-positive patients (defined by immunohistochemistry) treated with erlotinib and onartuzumab demonstrated improved progression-free and overall survival, whereas MET-negative patients had worse outcomes. This article provides an overview of signaling control by EGFR and MET and the biologic rationale for cotargeting strategies. EGFR and MET are membrane-bound RTKs consisting of extracellular domains that bind to growth factors and intracellular domains that contain a kinase domain (Fig 1). MET is activated by its cognate ligand, hepatocyte growth factor (HGF), which is produced by stromal cells, also in an autocrine fashion. EGFR is activated by several ligands, including EGF, transforming growth factor alpha (TGF ), and heparin-binding EGF. On receptor binding of the ligands, the kinase domain becomes activated, leading to transautophosphorylation of tyrosine residues on the cytoplasmic tail. Phosphorylation of these tyrosine residues creates intramolecular docking sites for proteins containing Src homology 2 domains that serve as readers for tyrosine phosphorylation. This includes adaptor proteins such as growth factor receptor–bound protein 2 (GRB2), GAB1 (GRB2-associated binding protein 1), and Src homology 2–containing transforming protein 1 (SHC1). These proteins serve as scaffolds to support signal transduction to the mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), phospholipase C (PLC), and signal transducer and activator of transcription (STAT) pathways. EGFR and MET use a highly overlapping repertoire of signaling adaptors and downstream effector pathways, highlighting their combined ability to codrive oncogenic signaling. Three general approaches are being employed in clinic to target EGFR and MET. The first uses small-molecule TKIs, such as erlotinib for EGFR and crizotinib for MET, that bind the ATP-binding pocket in the kinase domain and inhibit the kinase function. The second approach uses antibody therapy directed against the extracellular domain of the MET receptor (onartuzumab) or the HGF ligand (ficlatuzumab). The third approach targets downstream proteins involved in signaling, such as inhibitors of PI3K, or molecules regulating receptor trafficking and degradation, such as heat shock proteins. The EGFR pathway can be activated in lung cancer through genomic events, such as activating EGFR mutations or gene amplification, or through tumor environmental events, such as autocrine production of EGFR-activating ligands. Likewise, MET activation occurs through HGF produced in the tumor microenvironment and through MET gene amplification. MET amplification by fluorescent in situ hybridization occurs in approximately 5% to 15% of lung adenocarcinomas. Increased MET is associated with advanced stage of disease, poor outcome, brain metastasis, and high levels of EGFR. Coactivation of EGFR and MET occurs in subsets of lung cancer cell lines and tumor tissues and is associated with aggressive disease. In cells lacking EGFR mutation or high levels of MET, activation of either receptor can produce extensive signaling crosstalk to the opposing RTK and downstream substrates. EGFR activation can induce accumulation of activated MET protein through a c-SRC– dependent mechanism, and dual MET/EGFR inhibition has shown increased activity in xenograft models. Phosphoproteomic analysis of lung tumors and cell lines can identify tumors with concomitant EGFR and MET activation. EGFR, either through mutation or ligand-mediated activation, can also regulate MET levels through hypoxia-inducible factor-1 . Importantly, MET signaling is one actionable mechanism driving resistance to EGFR TKIs. MET activation can drive intrinsic as well as acquired resistance to EGFR through genomic and tumor microenvironment–mediated mechanisms. Primary resistance to anti-EGFR antibody therapy has been associated with MET activation in primary explant models of lung cancer. Approximately 5% of EGFR mutation–positive tumors with acquired resistance to EGFR TKIs are found to have MET gene amplification. Mechanistically, amplified MET co-opts use of ERBB3 to maintain the PI3K pathway activation. Combining MET and EGFR TKIs or EGFR/HSP90 inhibition has been effective in mouse models of lung cancer with T790M EGFR and MET amplification. High levels of HGF are associated with intrinsic or acquired resistance to EGFR TKIs in patients with lung cancer with activating EGFR mutations and in those with other solid tumors. HGF production by stromal fibroblasts leads to fibroblast-induced JOURNAL OF CLINICAL ONCOLOGY U N D E R S T A N D I N G T H E P A T H W A Y VOLUME 31 NUMBER 32 NOVEMBER 1

Astrocytic neoplasms show a high incidence of elevated or mutated epidermal growth factor receptor (EGFR) expression. Although proliferative effects from EGFR activation are well described, the role that changes in this receptor play in glioma growth and migration remain poorly addressed. This report characterizes changes in the levels of EGFR expression in three glial tumors at initial presentation (resection) and at the time of recurrence. By quantitative flow cytometry the mean level of EGFR expression increased, decreased, or remained the same in different recurrent astrocytomas relative to their primary tumor cells. Immunocytochemistry for EGFR on monolayer cells corroborated the level of expression in the recurrent tumors relative to their matched primary specimen. Immunoprecipitation indicated that 170 kd EGFR was expressed in each of the tumors, and showed normal down regulation following treatment with EGF. Proliferation response to EGF was seen in only 1/6 instances, but was concentration-dependent when observed. Stimulated migration of the cells was frequently seen and was also concentration-dependent on EGF; the magnitude of response was related to the relative level of 170 kd EGFR expression in the cells. EGFR immunostaining of tissue sections from the tumors confirmed the levels of EGFR expressed in primary and recurrent astrocytomas as was seen in the cultured cells. These results indicate that the relative levels of EGFR in early passage cell cultures from glioma specimens concurs with the measured tissue levels of expression. Human glioma cells are more responsive to migration induction than proliferation induction by EGF.

Selenoprotein W controls epidermal growth factor receptor surface expression, activation and degradation via receptor ubiquitination

Roles of mitogen activated protein kinases and EGF receptor in arsenite-stimulated matrix metalloproteinase-9 production

The activation of the epidermal growth factor receptor (EGFR) is crucial for triggering diverse cellular functions, including cell proliferation, migration, and differentiation, and up-regulation of EGFR expression or activity is a key factor in triggering the development of cancer. Here we show that overexpression of a scaffold protein, tumor necrosis factor receptor (TNF-R)-associated factor 4 (TRAF4), promotes EGF-induced autophosphorylation of EGFR (activation) and downstream signaling, whereas TRAF4 deficiency attenuates EGFR activation and EGF-driven cell proliferation. Using structure-based sequence alignment and NMR spectroscopy, we identified a TRAF4 binding site in the C-terminal half of the juxtamembrane (JM) segment of EGFR, a region known to promote asymmetric dimerization and subsequent activation. Deletion of the TRAF4 binding site led to dramatic defects in EGFR activation and EGF-driven cell proliferation. Specific point mutations in the TRAF4 binding site also resulted in significant attenuation of EGFR activation. Detailed structural examination of the inactive versus active forms of EGFR suggests that TRAF4 binding probably induces a conformational rearrangement of the JM region to promote EGFR dimerization. These results identify a novel mechanism of TRAF4-mediated EGFR activation and signaling.

Neu3 Activity Enhances Egfr Activation Without Affecting Egfr Expression

The mechanism of cleavage of EGFR ligands induced by inflammatory cytokines in gastric cancer cells

The ligand-mediated down-regulation of the growth factor receptors is preceded by the involvement of various other factors. In particular, a ubiquitin ligase, Cbl, plays a central role in this event. Several candidates that have potential effects on the negative control of the epidermal growth factor (EGF) receptor have now been identified by our recent studies in phospho-proteomics. Among these molecules, we focus on characterizing a novel protein, Ymer, which is a tyrosine-phosphorylated and ubiquitinated protein. Ymer is found to be phosphorylated at tyrosine 145 and 146 upon EGF stimulation, and lysine 129 of Ymer has been identified as a ubiquitination site. Ymer has two motifs interacting with the ubiquitin (MIU) domains that might function as a binding site for the ubiquitinated EGF receptor. Although Ymer and EGF receptors are associated in an EGF-dependent manner, their interaction is required not only for MIU domains but also for the tyrosine phosphorylation of Ymer. Phosphorylated Ymer is mainly located at the plasma membrane with EGF receptor and functions in its endocytosis and degradation. Furthermore, EGF-mediated secondary modifications of an activated-EGF receptor are inhibited by overexpressing Ymer in COS7 cells. Therefore, Ymer may have competitive effects on the activation of the EGF receptor. Our findings suggest that Ymer functions as a novel inhibitor for the down-regulation of the EGF receptor and plays a crucial role for regulating the amount of the EGF receptor on the cell surface membrane.

Aberrant epidermal growth factor receptor (EGFR) activation is observed in over 50% of cases of adult glioblastoma (GBM). Nevertheless, EGFR antibodies are ineffective in clinical GBM treatment, suggesting the existence of redundant EGFR activation mechanisms. Whether circular RNA (circRNA) encodes a protein involved in EGFR-driven GBM remains unclear. We reported an unexpected mechanism in which circular EGFR RNA (circ-EGFR) encodes a novel EGFR variant to sustained EGFR activation. We used RNA-seq, Northern blot, and Sanger sequencing to confirm the existence of circ-EGFR. Antibodies and a liquid chromatograph tandem mass spectrometer were used to identify circ-EGFR protein products. Lentivirus-transfected stable cell lines were used to assess the biological functions of the novel protein in vitro and in vivo. Clinical implications of circ-EGFR were assessed using 97 pathologically diagnosed GBM patient samples. The infinite open reading frame (iORF) in circ-EGFR translated repeating amino acid sequences via rolling translation and programmed -1 ribosomal frameshifting (-1PRF) induced out-of-frame stop codon (OSC), forming a polymetric novel protein-complex, which we termed rolling-translated EGFR (rtEGFR). rtEGFR directly interacted with EGFR, maintained EGFR membrane localization and attenuated EGFR endocytosis and degradation. Importantly, circ-EGFR levels correlated with the EGFR signature and predicted the poor prognosis of GBM patients. Deprivation of rtEGFR in brain tumor-initiating cells (BTICs) attenuated tumorigenicity and enhanced the anti-GBM effect. Our findings identified the endogenous rolling-translated protein and provided strong clinical evidence that targeting rtEGFR could improve the efficiency of EGFR-targeting therapies in GBM.

Epidermal growth factor (EGF) receptor (EGFR) is the founding member of the ErbB family of growth factor receptors that control growth, proliferation, differentiation, apoptosis, and cell migration. Selenoprotein W (SEPW1) is a highly conserved, ubiquitously expressed 9 kDa selenocysteine‐containing protein involved in cell cycle control. SEPW1 siRNA inhibited EGFR activation in RWPE‐1, MCF‐10A and MCF‐7 cells, while SEPW1 overexpression increased it. SEPW1 siRNA inhibited EGF‐induced activation of downstream pathways (MAPK, PI3K/Akt, JAK/STAT, and p53), and blocked EGF‐induced cell cycle entry. SEPW1 was associated with peripheral punctuate structures that partially co‐localized with EGFR. Ligand‐bound EGFR failed to traffic towards the nucleus in SEPW1‐depleted cells, indicating that SEPW1 has a role in EGFR trafficking. Consequently, the half‐life, expression, dimerization, and phosphorylation of EGFR were also decreased in SEPW1‐depleted cells. SEPW1 siRNA also inhibited hydrogen peroxide‐induced EGFR phosphorylation. However, SEPW1 siRNA inhibition of EGF‐stimulated EGFR phosphorylation was not decreased by N‐acetylcysteine or sodium orthovanadate, indicating SEPW1 regulation of EGFR does not involve reactive oxygen species or inhibition of protein tyrosine phosphatase. Glutathione peroxidase siRNA partially rescued EGF‐induced cell cycle entry in SEPW1‐depleted cells, showing the effect of SEPW1 depletion is not due to loss of antioxidant protection. Knowledge of the mechanisms underlying SEPW1 regulation of EGFR trafficking may be important to understanding the relationship between dietary selenium and cancer.Grant Funding Source: Supported by USDA CRIS Project 5306‐51530‐018‐00D

Many human epithelial cancers are characterized by abnormal activation of the epidermal growth factor receptor (EGFR), which is often caused by its excessive expression in tumor cells. The abundance of EGFR is modulated, in part, by its ubiquitination, which targets it for degradation. The components responsible for adding ubiquitin to EGFR are well characterized, but this is a reversible process, and the mechanisms that modulate the removal of ubiquitin from the EGFR are not well known. We found that de-ubiquitination of EGFR was regulated by diacylglycerol kinase delta (DGKdelta), a lipid kinase that terminates diacylglycerol signaling. In DGKdelta-deficient cells, ubiquitination of EGFR was enhanced, which attenuated the steady-state levels of EGFR and promoted its ligand-induced degradation. These effects were not caused by changes in the ubiquitinating apparatus, but instead were due to reduced expression of the de-ubiquitinase, ubiquitin-specific protease 8 (USP8). Depletion of protein kinase Calpha (PKCalpha), a target of diacylglycerol, rescued the levels of USP8 and normalized EGFR degradation in DGKdelta-deficient cells. Moreover, the effects of PKCalpha were caused by its inhibition of Akt, which stabilizes USP8. Our data indicate a novel mechanism where DGKdelta and PKCalpha modulate the levels of ubiquitinated EGFR through Akt and USP8.

The mechanisms underlying CD95 ligand (CD95L)- and hyperosmolarity-induced activation of the CD95 system [Reinehr, R., Graf, D., Fischer, R., Schliess, F., and Haussinger, D. (2002) Hepatology 36, 602-614] as initial steps of apoptosis were studied. Hyperosmotic exposure (405 mosmol/l) of rat hepatocytes induced within 1 min oxidative stress and antioxidant-sensitive activation of the epidermal growth factor receptor (EGFR) and c-Jun-N-terminal-kinase (JNK). After 30 min of hyperosmotic exposure EGFR associated with CD95 and CD95 became tyrosine phosphorylated. Inhibition of JNK or protein kinase C (PKC) had no effect on EGFR phosphorylation but abolished CD95/EGFR association, CD95-tyrosine phosphorylation, membrane targeting, and Fas-associated death domain/caspase 8 recruitment to CD95 [death-inducing signaling complex (DISC) formation]. Inhibition of EGFR tyrosine kinase activity prevented CD95 tyrosine phosphorylation and DISC formation but not hyperosmolarity-induced EGFR phosphorylation and EGFR association with CD95. Tyrosine-phosphorylated CD95 was enriched in the plasma membrane. All maneuvers preventing CD95 tyrosine phosphorylation inhibited CD95 membrane trafficking and DISC formation. Stimulation of EGFR by EGF induced EGFR phosphorylation but no association with CD95 or CD95 phosphorylation. Addition of CD95L also induced EGFR and JNK activation, EGFR/CD95 association, CD95 tyrosine phosphorylation, DISC formation, and CD95 membrane targeting with an inhibitor sensitivity profile similar to that of hyperosmotic CD95 activation, except that inhibition of PKC was ineffective. The data suggest that moderate hyperosmolarity or CD95L trigger oxidative stress and EGFR activation followed by a JNK-dependent EGFR/CD95association and CD95 tyrosine phosphorylation, probably through EGFR tyrosine kinase activity. This provides a signal for CD95 membrane trafficking and DISC formation.