Cellular localization of the activated EGFR determines its effect on cell growth in MDA-MB-468 cells.
ABSTRACT: The epidermal growth factor (EGF) receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase that regulates diverse cell functions that are dependent upon cell type, the presence of downstream effectors, and receptor density. In addition to activating biochemical pathways, ligand stimulation causes the EGFR to enter the cell via clathrin-coated pits. Endocytic trafficking influences receptor signaling by controlling the duration of EGFR phosphorylation and coordinating the receptor's association with downstream effectors. To better understand the individual contributions of cell surface and cytosolic EGFRs on cell physiology, we used EGF that was conjugated to 900 nm polystyrene beads (EGF-beads). EGF-beads can stimulate the EGFR and retain the activated receptor at the plasma membrane. In MDA-MB-468 cells, a breast cancer cell line that over-expresses the EGFR, only internalized, activated EGFRs stimulate caspase-3 and induce cell death. Conversely, signaling cascades triggered from activated EGFR retained at the cell surface inhibit caspase-3 and promote cell proliferation. Thus, through endocytosis, the activated EGFR can differentially regulate cell growth in MDA-MB-468 cells.
Project description:Many solid tumors, including breast cancer, show increased activation of several growth factor receptors, specifically epidermal growth factor receptor (EGFR) and its family members as well as c-Src, a nonreceptor tyrosine kinase that promotes proliferation, inhibits apoptosis, and induces metastasis. We hypothesize that inhibition of c-Src and EGFRs will be an effective therapeutic strategy for triple-negative breast cancer. To test our hypothesis, we used a c-Src-specific inhibitor dasatinib (BMS-354825; Bristol-Myers Squibb) and our newly developed ErbB-inhibitory protein (EBIP), a potential pan-ErbB inhibitor, in breast cancer cells. EBIP is composed of 1 to 448 amino acids of the ectodomain of human EGFR to which the 30-amino acid epitope (known as "U" region) of rat EGFR-related protein is fused at the COOH-terminal end. The combination of dasatinib and EBIP was found to be highly effective in inhibiting the growth of four different breast cancer cells (MDA-MB-468, SKBr-3, MDA-MB-453, and MDA-MB-231) that express different levels of EGFRs. In EGFR-overexpressing MDA-MB-468 cells, the combination, but not monotherapy, markedly stimulated apoptosis mediated by caspase-9 and caspase-8 and attenuated activation of EGFR and Src as well as tyrosine kinase activity. EBIP also inhibited heregulin-induced activation of HER-2 and HER-3 in MDA-MB-453 breast cancer cells. The combination therapy was highly effective in suppressing tumor growth ( approximately 90% inhibition) in MDA-MB-468-derived xenografts in severe combined immunodeficient mice. The latter could be attributed to induction of apoptosis. We conclude that combining dasatinib and EBIP could be an effective therapeutic strategy for breast cancer by targeting EGFRs and Src signaling.
Project description:Activation of epidermal growth factor (EGF) receptor (EGFR) leads to its interaction with Grb2, a dual-function adapter mediating both signaling through Ras and receptor endocytosis. We used time-lapse three-dimensional imaging by spinning disk confocal microscopy to analyze trafficking of EGFR and Grb2 in living HeLa cells stimulated with low, physiological concentrations of EGFR ligands. Endogenous Grb2 was replaced in these cells by Grb2 fused to yellow fluorescent protein (YFP). After transient residence in the plasma membrane, Rhodamine-conjugated EGF (EGF-Rh) and Grb2-YFP were rapidly internalized and accumulated in endosomes. Quantitative image analysis revealed that on average two Grb2-YFP molecules were colocalized with one EGF-Rh in cells stimulated with 2 ng/ml EGF-Rh, and the excess of Grb2-YFP over EGF-Rh was even higher when a receptor-saturating concentration of EGF-Rh was used. Therefore, we hypothesize that a single EGFR molecule can be simultaneously associated with functionally distinct Grb2 interaction partners during and after endocytosis. Continuous presence of Grb2-YFP in endosomes was also observed when EGFR was activated by transforming growth factor-α and amphiregulin, suggesting that endosomal EGFRs remain ligand occupied and signaling competent, despite the fact that these growth factors are thought to dissociate from the receptor at acidic pH. The prolonged localization and activity of EGFR-Grb2 complexes in endosomes correlated with the sustained activation of extracellular stimulus-regulated kinase 1/2, suggesting that endosomal EGFRs contribute significantly to this signaling pathway. We propose that endosomal EGFRs function to extend signaling in time and space to compensate for rapid downregulation of surface EGFRs in cells with low receptor expression levels.
Project description:Nanomedicines allow active targeting of cancer for diagnostic and therapeutic applications through incorporation of multiple functional components. Frequently, however, clinical translation is hindered by poor intratumoural delivery and distribution. The application of physical stimuli to promote tumour uptake is a viable route to overcome this limitation. In this study, ultrasound-mediated cavitation of microbubbles was investigated as a mean of enhancing the delivery of a liposome designed for chemo-radionuclide therapy targeted to EGFR overexpressing cancer. Method: Liposomes (111In-EGF-LP-Dox) were prepared by encapsulation of doxorubicin (Dox) and surface functionalisation with Indium-111 tagged epidermal growth factor. Human breast cancer cell lines with high and low EGFR expression (MDA-MB-468 and MCF7 respectively) were used to study selectivity of liposomal uptake, subcellular localisation of drug payload, cytotoxicity and DNA damage. Liposome extravasation following ultrasound-induced cavitation of microbubbles (SonoVue®) was studied using a tissue-mimicking phantom. In vivo stability, pharmacokinetic profile and biodistribution were evaluated following intravenous administration of 111In-labelled, EGF-functionalised liposomes to mice bearing subcutaneous MDA-MB-468 xenografts. Finally, the influence of ultrasound-mediated cavitation on the delivery of liposomes into tumours was studied. Results: Liposomes were loaded efficiently with Dox, surface decorated with 111In-EGF and showed selective uptake in MDA-MB-468 cells compared to MCF7. Following binding to EGFR, Dox was released into the intracellular space and 111In-EGF shuttled to the cell nucleus. DNA damage and cell kill were higher in MDA-MB-468 than MCF7 cells. Moreover, Dox and 111In were shown to have an additive cytotoxic effect in MDA-MB-468 cells. US-mediated cavitation increased the extravasation of liposomes in an in vitro gel phantom model. In vivo, the application of ultrasound with microbubbles increased tumour uptake by 66% (p<0.05) despite poor vascularisation of MDA-MB-468 xenografts (as shown by DCE-MRI). Conclusion: 111In-EGF-LP-Dox designed for concurrent chemo-radionuclide therapy showed specificity for and cytotoxicity towards EGFR-overexpressing cancer cells. Delivery to tumours was enhanced by the use of ultrasound-mediated cavitation indicating that this approach has the potential to deliver cytotoxic levels of therapeutic radionuclide to solid tumours.
Project description:The Epidermal Growth Factor Receptor (EGFR) is a transmembrane receptor tyrosine kinase with critical implications in cell proliferation, migration, wound healing and the regulation of apoptosis. However, the EGFR has been shown to be hyper-expressed in a number of human malignancies. The MDA-MB-468 metastatic breast cell line is one example of this. This particular cell line hyper-expresses the EGFR and undergoes EGFR-mediated apoptosis in response to EGF ligand. The goal of this study was to identify the kinases that could be potential intermediates for the EGFR-mediated induction of apoptosis intracellularly. After identifying Cyclic GMP-dependent Protein Kinase G (PKG) as a plausible intermediate, we wanted to determine the temporal relationship of these two proteins in the induction of apoptosis. We observed a dose-dependent decrease in MDA-MB-468 cell viability, which was co-incident with increased PKG activity as measured by VASPSer239 phosphorylation. In addition, we observed a dose dependent decrease in cell viability, as well as an increase in apoptosis, in response to two different PKG agonists, 8-Bromo-cGMP and 8-pCPT-cGMP. MDA-MB-468 cells with reduced PKG activity had attenuated EGFR-mediated apoptosis. These findings indicate that PKG does not induce cell death via transphosphorylation of the EGFR. Instead, PKG activity occurs following EGFR activation. Together, these data indicate PKG as an intermediary in EGFR-mediated cell death, likely via apoptotic pathway.
Project description:Purpose Radiolabeled antibodies and peptides hold promise for molecular radiotherapy but are often limited by a low payload resulting in inadequate delivery of radioactivity to tumour tissue and, therefore, modest therapeutic effect. We developed a facile synthetic method of radiolabeling indium-111 (111In) to epidermal growth factor (EGF)-gold nanoparticles (111In-EGF-Au NP) with a high payload. Materials and methods EGF-Au NP were prepared via an interaction between gold and the disulphide bonds of EGF and radiolabeled using 111InCl3. Targeting efficiency was investigated by quantitating internalized radioactivity and by confocal imaging following exposure of MDA-MB-468 (1.3 × 106 EGFR/cell) and MCF-7 (104 EGFR/cell) cells to Cy3-EGF-Au NP. Cytotoxicity was evaluated in clonogenic assays. Results The proportion of total administered radioactivity that was internalized by MDA-MB-468 and MCF-7 cells was 15% and 1.3%, respectively (mixing ratio of EGF:Au of 160). This differential uptake in the two cell lines was confirmed using confocal microscopy. 111In-EGF-Au NP were significantly more radiotoxic to MDA-MB-468 than MCF-7 cells with a surviving fraction of 17.1 ± 4.4% versus 89.8 ± 1.4% (p < 0.001) after exposure for 4?h. Conclusions An 111In-labeled EGF-Au nanosystem was developed. It enabled targeted delivery of a high 111In payload specifically to EGFR-positive cancer cells leading to radiotoxicity that can be exploited for molecularly targeted radiotherapy.
Project description:Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) that controls cell proliferation, growth, survival, metabolism, and migration by activating the PI3K (phosphatidylinositol 3-kinase)-AKT and ERK (extracellular signal-regulated kinase)-RSK (ribosomal S6 kinase) pathways. EGFR signaling to these pathways is temporally and spatially regulated. Endocytic trafficking controls the access of EGFR to these downstream effectors and also its degradation, which terminates EGFR signaling. We showed that AKT facilitated the endocytic trafficking of EGFR to promote its degradation. Interfering with AKT signaling reduced both EGFR recycling and the rate of EGFR degradation. In AKT-impaired cells, EGFRs were unable to reach the cell surface or the lysosomal compartment and accumulated in the early endosomes, resulting in prolonged signaling and increased activation of ERK and RSK. Upon EGF stimulation, AKT phosphorylated and activated the kinase PIKfyve [FYVE-containing phosphatidylinositol 3-phosphate 5-kinase], which promoted vesicle trafficking to lysosomes. PIKfyve activation promoted EGFR degradation. Similar regulation occurred with platelet-derived growth factor receptor (PDGFR), suggesting that AKT phosphorylation and activation of PIKfyve is likely to be a common feedback mechanism for terminating RTK signaling and reducing receptor abundance.
Project description:Activated epidermal growth factor receptors (EGFRs) are crucial for inducing metastasis in cancer cells by promoting matrix metalloproteinase (MMP) expression. The present study was designed to investigate the effects of 1?palmitoyl?2?linoleoyl?3?acetyl?rac?glycerol (PLAG) on MMP expression in epidermal growth factor (EGF)?stimulated breast cancer cells in vitro. EGF stimulation induced internalization of its cognate receptor, EGFR, for stimulus?desensitization. These internalized receptors, complexed with the ubiquitin ligase c?Cbl and EGFR pathway substrate 15 (EPS15) (for degradation), were evaluated by confocal microscopy at 5?90 min time intervals. During intracellular trafficking of EGFRs, EGF?induced signaling cascades were analyzed by examining EGFR and SHC phosphorylation. Modulation of MMP expression was assessed by evaluating the activity of transcription factor AP?1 using a luciferase assay. PLAG accelerated the assembly of EGFRs with c?Cbl and EPS15 and promoted receptor degradation. This faster intracellular EGFR degradation reduced AP?1?mediated MMP expression. PLAG stimulation upregulated thioredoxin?interacting protein (TXNIP) expression, and this mediated the accelerated receptor internalization. This PLAG?induced increase in EGFR trafficking was blocked in TXNIP?silenced cells. By downregulating MMP expression, PLAG effectively attenuated EGF?induced mobility and invasiveness in these cancer cells. These data suggest that PLAG may be a potential therapeutic agent for blocking metastasis.
Project description:Breast cancers that express epidermal growth factor (EGF) receptors (EGFRs) are associated with poor prognosis. Our group recently showed in breast cancer patients that EGFR expression is strongly correlated with high tumor uptake of the glucose analogue, 18F-fluorodeoxyglucose (FDG). Here, we explored the cellular mechanism and signaling pathways that can explain the relation between EGFR and breast cancer cell glucose metabolism. FDG uptake, lactate production and hexokinase (HK) activity were measured, and proliferation assays and western blots were performed. EGF stimulated an increase of FDG uptake in EGFR-positive T47D and MDA-MB-468 cells, but not in MCF-7 cells. In T47D cells, the effect was dose-dependent and was accompanied by increased lactate production, indicating a shift toward glycolytic flux. This metabolic response occurred through enhanced HK activity and upregulated glucose transporter 1 (GLUT1) expression. EGFR stimulation also increased T47D cell proliferation. Blocking EGFR activation with BIBX1382 or gefitinib completely abolished both FDG uptake and proliferation effects. EGFR stimulation induced MAP kinase (MAPK) and PI3 kinase (PI3K) activation. Increased cell proliferation by EGFR stimulation was completely abolished by MAPK inhibition with PD98059 or by PI3K inhibition with LY294002. Increased FDG uptake was also completely abrogated by PI3K inhibition but was uninfluenced by MAPK inhibition. These findings suggest that the association between breast tumor EGFR expression and high FDG uptake might be contributed by stimulation of the PI3K pathway downstream of EGFR activation. This was in contrast to EGFR-mediated cell proliferation that required MAPK as well as PI3K signaling.
Project description:Previously, it was shown that a novel 4-(N)-stearoyl gemcitabine nanoparticle formulation was more effective than gemcitabine hydrochloride in controlling the growth of model mouse or human tumors pre-established in mice. In the present study, the feasibility of targeting the stearoyl gemcitabine nanoparticles (GemC18-NPs) into tumor cells that over-express epidermal growth factor receptor (EGFR) to more effectively control tumor growth was evaluated. EGFR is over-expressed in a variety of tumor cells, and EGF is a known natural ligand of EGFR. Recombinant murine EGF was conjugated onto the GemC18-NPs. The ability of the EGF to target the GemC18-NPs to human breast adenocarcinoma cells that expressed different levels of EGFR was evaluated in vitro and in vivo. In culture, the extent to which the EGF-conjugated GemC18-NPs were taken up by tumor cells was correlated to the EGFR density on the tumor cells, whereas the uptake of untargeted GemC18-NPs exhibited no difference among those same cell lines. The relative cytotoxicity of the EGF-conjugated GemC18-NPs to tumor cells in culture was correlated to EGFR expression as well. In vivo, EGFR-over-expressing MDA-MB-468 tumors in mice treated with the EGF-conjugated GemC18-NPs grew significantly slower than in mice treated with untargeted GemC18-NPs, likely due to that the EGF-GemC18-NPs were more anti-proliferative, anti-angiogenic, and pro-apoptotic. Fluorescence intensity data from ex vivo imaging showed that the EGF on the nanoparticles helped increase the accumulation of the GemC18-NPs into MDA-MB-468 tumors pre-established in mice by more than 2-fold as compared to the un-targeted GemC18-NPs. In conclusion, active targeting of the GemC18-NPs into EGFR-over-expressed tumors can further enhance their anti-tumor activity.
Project description:Multivalent presentation of ligands on nanoparticles (NPs) is considered a general strategy for enhancing receptor binding and activation through amplification of ligand-receptor interactions within the footprint of the individual NPs. The spatial clustering of ligand-functionalized NPs represents an additional, less well understood mechanism for increasing local ligand-receptor interactions, especially for receptors that form higher-order assemblies, such as the epidermal growth factor (EGF) receptor (EGFR). To shed light on the interplay between ligand density ( i.e., multivalency) and NP clustering in signal amplification, we apply EGF-functionalized 72 ± 1 nm gold nanoparticles (NP-EGF) with known ligand loading (10-200 EGF/NP) as quantifiable and experimentally tractable units of EGFR activation and characterize the NP-mediated amplification of EGFR phosphorylation as a function of both EGF surface density and NP-EGF clustering for two cancer cell lines (HeLa and MDA-MB-468). The measurements confirm a strong multivalent amplification of EGFR phosphorylation through NP-EGF on the cellular level that results in EGF-loading-dependent maximum EGFR phosphorylation levels. A microscopic analysis of NP-EGF-induced EGFR phosphorylation reveals a heterogeneous spatial distribution of EGFR activation across the cell surface. Clustering of multivalent NP-EGF on sub-diffraction-limited length scales is found to result in a local enhancement of EGFR phosphorylation in signaling "hot spots" from where the signal can spread laterally in an EGF-independent fashion. Increasing EGF loadings of the NP enhances NP-EGF clustering and intensifies EGFR phosphorylation. These observations suggest that NP-EGF clustering and the associated local enhancement of ligand-receptor interactions are intrinsic components of the multivalent amplification of phosphorylation for the heterogeneously distributed EGFR through NP-EGF.