EGFRvIII uses intrinsic and extrinsic mechanisms to reduce glioma adhesion and increase migration

Afsheen Banisadr; Mariam Eick; Pranjali Beri; Alison D. Parisian; Benjamin Yeoman; Jesse K. Placone; Adam J. Engler; Frank Furnari

doi:10.1242/jcs.247189

EGFRvIII uses intrinsic and extrinsic mechanisms to reduce glioma adhesion and increase migration

Journal of Cell Science ◽

10.1242/jcs.247189 ◽

2020 ◽

Vol 133 (24) ◽

pp. jcs247189

Author(s):

Afsheen Banisadr ◽

Mariam Eick ◽

Pranjali Beri ◽

Alison D. Parisian ◽

Benjamin Yeoman ◽

...

Keyword(s):

Glioblastoma Multiforme ◽

Focal Adhesion ◽

Biological Markers ◽

Egf Receptor ◽

Receptor Kinase ◽

Wild Type ◽

Invasive Potential ◽

Intrinsic Signals ◽

Surrounding Parenchyma ◽

Constitutively Active

ABSTRACTA lack of biological markers has limited our ability to identify the invasive cells responsible for glioblastoma multiforme (GBM). To become migratory and invasive, cells must downregulate matrix adhesions, which could be a physical marker of invasive potential. We engineered murine astrocytes with common GBM mutations, e.g. Ink4a (Ink) or PTEN deletion and expressing a constitutively active EGF receptor truncation (EGFRvIII), to elucidate their effect on adhesion. While loss of Ink or PTEN did not affect adhesion, counterparts expressing EGFRvIII were significantly less adhesive. EGFRvIII reduced focal adhesion size and number, and these cells – with more labile adhesions – displayed enhanced migration. Regulation appears to depend not on physical receptor association to integrins but, rather, on the activity of the receptor kinase, resulting in transcriptional integrin repression. Interestingly, EGFRvIII intrinsic signals can be propagated by cytokine crosstalk to cells expressing wild-type EGFR, resulting in reduced adhesion and enhanced migration. These data identify potential intrinsic and extrinsic mechanisms that gliomas use to invade surrounding parenchyma.

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STIM1 Controls the Focal Adhesion Dynamics and Cell Migration by Regulating SOCE in Osteosarcoma

International Journal of Molecular Sciences ◽

10.3390/ijms23010162 ◽

2021 ◽

Vol 23 (1) ◽

pp. 162

Author(s):

Yu-Shan Lin ◽

Yi-Hsin Lin ◽

MyHang Nguyen Thi ◽

Shih-Chuan Hsiao ◽

Wen-Tai Chiu

Keyword(s):

Cell Migration ◽

Focal Adhesion ◽

Cancer Metastasis ◽

Focal Adhesions ◽

Dominant Negative ◽

Osteosarcoma Cell ◽

Wild Type ◽

Calpain Activity ◽

The Impact ◽

Constitutively Active

The dysregulation of store-operated Ca2+ entry (SOCE) promotes cancer progression by changing Ca2+ levels in the cytosol or endoplasmic reticulum. Stromal interaction molecule 1 (STIM1), a component of SOCE, is upregulated in several types of cancer and responsible for cancer cell migration, invasion, and metastasis. To explore the impact of STIM1-mediated SOCE on the turnover of focal adhesion (FA) and cell migration, we overexpressed the wild-type and constitutively active or dominant negative variants of STIM1 in an osteosarcoma cell line. In this study, we hypothesized that STIM1-mediated Ca2+ elevation may increase cell migration. We found that constitutively active STIM1 dramatically increased the Ca2+ influx, calpain activity, and turnover of FA proteins, such as the focal adhesion kinase (FAK), paxillin, and vinculin, which impede the cell migration ability. In contrast, dominant negative STIM1 decreased the turnover of FA proteins as its wild-type variant compared to the cells without STIM1 overexpression while promoting cell migration. These unexpected results suggest that cancer cells need an appropriate amount of Ca2+ to control the assembly and disassembly of focal adhesions by regulating calpain activity. On the other hand, overloaded Ca2+ results in excessive calpain activity, which is not beneficial for cancer metastasis.

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947: Chemopreventive Effects of COX-2 Inhibitor and Epidermal Growth Factor (EGF)-Receptor Kinase Inhibitor on Rat Urinary Bladder Tumorigenesis

The Journal of Urology ◽

10.1016/s0022-5347(18)38184-9 ◽

2004 ◽

Vol 171 (4S) ◽

pp. 251-251

Author(s):

Kazunori Hattori ◽

Katsuyuki Iida ◽

Akira Johraku ◽

Sadamu Tsukamoto ◽

Taeko Asano ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Urinary Bladder ◽

Egf Receptor ◽

Kinase Inhibitor ◽

Receptor Kinase ◽

Rat Urinary Bladder ◽

Epidermal Growth ◽

Cox 2

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Photoaffinity labelling of the ATP-binding site of the epidermal growth factor-dependent protein kinase

Biochemical Journal ◽

10.1042/bj2200677 ◽

1984 ◽

Vol 220 (3) ◽

pp. 677-683 ◽

Cited By ~ 6

Author(s):

J E Kudlow ◽

Y Leung

Keyword(s):

Protein Kinase ◽

Binding Site ◽

Egf Receptor ◽

Light Exposure ◽

Atp Binding ◽

Receptor Kinase ◽

Dependent Protein Kinase ◽

Atp Binding Site ◽

Dependent Protein ◽

Epidermal Growth

Epidermal growth factor (EGF), after binding to its receptor, activates a tyrosine-specific protein kinase which phosphorylates several substrates, including the EGF receptor itself. The effects of a photoaffinity analogue of ATP, 3′-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)adenosine 5′-triphosphate (arylazido-beta-alanyl-ATP) on the EGF-dependent protein kinase in A431 human tumour cell plasma membrane vesicles was investigated. This analogue was capable of inactivating the EGF-receptor kinase in a photodependent manner. Partial inactivation occurred at an analogue concentration of 1 microM and complete inactivation occurred at 10 microM when a 2 min light exposure was used. Arylazido-beta-alanine at 100 microM and ATP at 100 microM were incapable of inactivating the enzyme with 2 min of light exposure. The photodependent inactivation of the enzyme by the analogue could be partially blocked by 20 mM-ATP and more effectively blocked by either 20 mM-adenosine 5′-[beta gamma-imido]triphosphate or 20 mM-guanosine 5′-[beta gamma-imido]triphosphate, indicating nucleotide-binding site specificity. Arylazido-beta-alanyl-[alpha-32P]ATP was capable of labelling membrane proteins in a photodependent manner. Numerous proteins were labelled, the most prominent of which ran with an apparent Mr of 53000 on polyacrylamide-gel electrophoresis. A band of minor intensity was seen of Mr corresponding to the EGF receptor (170000). Immunoprecipitation of affinity-labelled and solubilized membranes with an anti-(EGF receptor) monoclonal antibody demonstrated that the Mr 170000 receptor protein was photoaffinity labelled by the analogue. The Mr 53000 peptide was not specifically bound by the anti-receptor antibody. The affinity labelling of the receptor was not enhanced by EGF, suggesting that EGF stimulation of the kinase activity does not result from changes in the affinity of the kinase for ATP. These studies demonstrate that arylazido-beta-alanyl-ATP interacts with the ATP-binding site of the EGF-receptor kinase with apparent high affinity and that this analogue is an effective photoaffinity label for the kinase. Furthermore, these studies demonstrate that the EGF receptor, identified by using monoclonal antibodies, contains an ATP-binding site, providing further confirmation that the EGF receptor and EGF-dependent protein kinase are domains of the Mr 170000 protein.

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Characteristics and uses of antibodies to the EGF-receptor kinase

Cell Biology International Reports ◽

10.1016/0309-1651(83)90162-5 ◽

1983 ◽

Vol 7 (7) ◽

pp. 529-530 ◽

Cited By ~ 6

Author(s):

C STOSCHECK ◽

G CARPENTER

Keyword(s):

Egf Receptor ◽

Receptor Kinase

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Activation and inhibition of erythropoietin receptor function: role of receptor dimerization

Molecular and Cellular Biology ◽

10.1128/mcb.14.6.3535-3549.1994 ◽

1994 ◽

Vol 14 (6) ◽

pp. 3535-3549

Author(s):

S S Watowich ◽

D J Hilton ◽

H F Lodish

Keyword(s):

Signal Transduction ◽

Growth Hormone Receptor ◽

Initial Step ◽

Inhibitory Effect ◽

Erythropoietin Receptor ◽

Interface Region ◽

Wild Type ◽

Receptor Dimerization ◽

Dimer Interface ◽

Constitutively Active

Members of the cytokine receptor superfamily have structurally similar extracellular ligand-binding domains yet diverse cytoplasmic regions lacking any obvious catalytic domains. Many of these receptors form ligand-induced oligomers which are likely to participate in transmembrane signaling. A constitutively active (factor-independent) mutant of the erythropoietin receptor (EPO-R), R129C in the exoplasmic domain, forms disulfide-linked homodimers, suggesting that the wild-type EPO-R is activated by ligand-induced homodimerization. Here, we have taken two approaches to probe the role EPO-R dimerization plays in signal transduction. First, on the basis of the crystal structure of the ligand-bound, homodimeric growth hormone receptor (GH-R) and sequence alignment between the GH-R and EPO-R, we identified residues of the EPO-R which may be involved in intersubunit contacts in an EPO-R homodimer. Residue 129 of the EPO-R corresponds to a residue localized to the GH-R dimer interface region. Alanine or cysteine substitutions were introduced at four other residues of the EPO-R predicted to be in the dimer interface region. Substitution of residue E-132 or E-133 with cysteine renders the EPO-R constitutively active. Like the arginine-to-cysteine mutation at position 129 in the exoplasmic domain (R129C), E132C and E133C form disulfide-linked homodimers, suggesting that constitutive activity is due to covalent dimerization. In the second approach, we have coexpressed the wild-type EPO-R with inactive mutants of the receptor missing all or part of the cytosolic domain. These truncated receptors have a dominant inhibitory effect on the proliferative action of the wild-type receptor. Taken together, these results strengthen the hypothesis that an initial step in EPO- and EPO-R-mediated signal transduction is ligand-induced receptor dimerization.

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EGF receptor regulation of cell motility: EGF induces disassembly of focal adhesions independently of the motility-associated PLCgamma signaling pathway

Journal of Cell Science ◽

10.1242/jcs.111.5.615 ◽

1998 ◽

Vol 111 (5) ◽

pp. 615-624 ◽

Cited By ~ 8

Author(s):

H. Xie ◽

M.A. Pallero ◽

K. Gupta ◽

P. Chang ◽

M.F. Ware ◽

...

Keyword(s):

Growth Factor ◽

Cell Motility ◽

Map Kinase ◽

Signaling Pathway ◽

Focal Adhesion ◽

Kinase Activity ◽

Egf Receptor ◽

Focal Adhesions ◽

Short Term ◽

Egfr Kinase

A current model of growth factor-induced cell motility invokes integration of diverse biophysical processes required for cell motility, including dynamic formation and disruption of cell/substratum attachments along with extension of membrane protrusions. To define how these biophysical events are actuated by biochemical signaling pathways, we investigate here whether epidermal growth factor (EGF) induces disruption of focal adhesions in fibroblasts. We find that EGF treatment of NR6 fibroblasts presenting full-length WT EGF receptors (EGFR) reduces the fraction of cells presenting focal adhesions from approximately 60% to approximately 30% within 10 minutes. The dose dependency of focal adhesion disassembly mirrors that for EGF-enhanced cell motility, being noted at 0.1 nM EGF. EGFR kinase activity is required as cells expressing two kinase-defective EGFR constructs retain their focal adhesions in the presence of EGF. The short-term (30 minutes) disassembly of focal adhesions is reflected in decreased adhesiveness of EGF-treated cells to substratum. We further examine here known motility-associated pathways to determine whether these contribute to EGF-induced effects. We have previously demonstrated that phospholipase C(gamma) (PLCgamma) activation and mobilization of gelsolin from a plasma membrane-bound state are required for EGFR-mediated cell motility. In contrast, we find here that short-term focal adhesion disassembly is induced by a signaling-restricted truncated EGFR (c'973) which fails to activate PLCgamma or mobilize gelsolin. The PLC inhibitor U73122 has no effect on this process, nor is the actin severing capacity of gelsolin required as EGF treatment reduces focal adhesions in gelsolin-devoid fibroblasts, further supporting the contention that focal adhesion disassembly is signaled by a pathway distinct from that involving PLCgamma. Because both WT and c'973 EGFR activate the erk MAP kinase pathway, we additionally explore here this signaling pathway, not previously associated with growth factor-induced cell motility. Levels of the MEK inhibitor PD98059 that block EGF-induced mitogenesis and MAP kinase phosphorylation also abrogate EGF-induced focal adhesion disassembly and cell motility. In summary, we characterize for the first time the ability of EGFR kinase activity to directly stimulate focal adhesion disassembly and cell/substratum detachment, in relation to its ability to stimulate migration. Furthermore, we propose a model of EGF-induced motogenic cell responses in which the PLCgamma pathway stimulating cell motility is distinct from the MAP kinase-dependent signaling pathway leading to disassembly and reorganization of cell-substratum adhesion.

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Effect of Deletion of the Ribonucleotide Reductase Gene in Wild Type and Virion Associated Host Shutoff (vhs-1) Mutant Herpes Simplex Virus-1 on Viral Proliferation and Infected-Carcinoma Cell Cultures Growth

10.1101/2020.12.18.423438 ◽

2020 ◽

Author(s):

Pnina Schlesinger ◽

Niza Frenkel

Keyword(s):

Glioblastoma Multiforme ◽

Cell Cultures ◽

Ribonucleotide Reductase ◽

Viral Vector ◽

Green Fluorescence Protein ◽

Vero Cells ◽

Wild Type ◽

Herpes Simplex Virus 1 ◽

Host Shutoff ◽

Simplex Virus

Glioblastoma multiforme is the most prevalent and deadliest form of glioma and brain cancer, with a very poor prognosis. In an effort to develop an oncolytic viral vector for the treatment of Glioblastoma multiforme, we replaced the UL39 and UL40 genes encoding ribonucleotide reductase (RR) with green fluorescence protein and luciferase genes in wild type KOS and in the virion host shutoff mutant vhs-1, resulting in strains KOS-RR and Vhs-RR, respectively. KOS-RR and Vhs-RR caused death of infected U87 Glioblastoma multiforme cell cultures within one day after infection, whereas KOS and vhs-1-infected cells were more viable. All four viral strains caused apoptotic DNA laddering in infected H1299 lung cancer cells, while only Vhs-RR caused apoptosis in U87 cell cultures. Vhs-RR gave higher yields on U87 than on Vero cells, while it barely proliferated on non-dividing Goiter cells. These results indicate that Vhs-RR proliferates well in actively growing U87 Glioblastoma multiforme cells, causing their death in a mechanism involving apoptosis, while sparing non-dividing cells. Therefore, Vhs-RR is a promising candidate for oncolytic treatment of brain tumor malignancies.

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Could erlotinib be used in EGF receptor wild-type non-small-cell lung cancer?

Lung Cancer Management ◽

10.2217/lmt.13.39 ◽

2013 ◽

Vol 2 (5) ◽

pp. 333-336

Author(s):

Haiying Cheng ◽

Bilal Piperdi

Keyword(s):

Lung Cancer ◽

Small Cell Lung Cancer ◽

Cell Lung Cancer ◽

Egf Receptor ◽

Small Cell ◽

Wild Type ◽

Small Cell Lung

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Localization of Phospholipase D1 to Caveolin-enriched Membrane via Palmitoylation: Implications for Epidermal Growth Factor Signaling

Molecular Biology of the Cell ◽

10.1091/mbc.e02-02-0100 ◽

2002 ◽

Vol 13 (11) ◽

pp. 3976-3988 ◽

Cited By ~ 40

Author(s):

Jung Min Han ◽

Yong Kim ◽

Jun Sung Lee ◽

Chang Sup Lee ◽

Byoung Dae Lee ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Egf Receptor ◽

Point Mutations ◽

Dominant Negative ◽

Growth Factor Signaling ◽

Wild Type ◽

Caveolin 1 ◽

Epidermal Growth ◽

Egf Signaling

Phospholipase D (PLD) has been suggested to mediate epidermal growth factor (EGF) signaling. However, the molecular mechanism of EGF-induced PLD activation has not yet been elucidated. We investigated the importance of the phosphorylation and compartmentalization of PLD1 in EGF signaling. EGF treatment of COS-7 cells transiently expressing PLD1 stimulated PLD1 activity and induced PLD1 phosphorylation. The EGF-induced phosphorylation of threonine147 was completely blocked and the activity of PLD1 attenuated by point mutations (S2A/T147A/S561A) of PLD1 phosphorylation sites. The expression of a dominant negative PKCα mutant by adenovirus-mediated gene transfer greatly inhibited the phosphorylation and activation of PLD1 induced by EGF in PLD1-transfected COS-7 cells. EGF-induced PLD1 phosphorylation occurred primarily in the caveolin-enriched membrane (CEM) fraction, and the kinetics of PLD1 phosphorylation in the CEM were strongly correlated with PLD1 phosphorylation in the total membrane. Interestingly, EGF-induced PLD1 phosphorylation and activation and the coimmunoprecipitation of PLD1 with caveolin-1 and the EGF receptor in the CEM were significantly attenuated in the palmitoylation-deficient C240S/C241S mutant, which did not localize to the CEM. Immunocytochemical analysis revealed that wild-type PLD1 colocalized with caveolin-1 and the EGF receptor and that phosphorylated PLD1 was localized exclusively in the plasma membrane, although some PLD1 was also detected in vesicular structures. Transfection of wild-type PLD1 but not of C240S/C241S mutant increased EGF-induced raf-1 translocation to the CEM and ERK phosphorylation. This study shows, for the first time, that EGF-induced PLD1 phosphorylation and activation occur in the CEM and that the correct localization of PLD1 to the CEM via palmitoylation is critical for EGF signaling.

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